Uniformly moist cheese

ABSTRACT

The invention provides moist cheeses of uniform composition that are readily and inexpensively made by acidifying milk prior to beginning the cheese making process.

This application is a continuation of International Application No.PCT/US2006/034117 filed Aug. 30, 2006, which claims benefit of thefiling dates of U.S. Provisional Application Ser. No. 60/712,621, filedAug. 30, 2005, and U.S. Provisional Application Ser. No. 60/775,049,filed Feb. 20, 2006, the contents of which applications are incorporatedherein in their entireties.

FIELD OF THE INVENTION

The invention relates to methods for making blocks of cheese, where thecheese has improved moisture content and composition. For example, thecheese is uniformly moist throughout even large blocks of cheese. Thecheese also has an increased moisture content to prevent drying, improveshelf life and reduce manufacturing wastes and costs.

BACKGROUND OF THE INVENTION

In the United States, Cheddar cheese was traditionally produced in 18 kg(40 lb) blocks. In a highly cost-competitive market, more automated andefficient means of handling large quantities of cheese in rapidlyexpanding cheese factories were developed to control costs. Thus, in thelate 1970s and early 1980s, the first 290 kg (640 lb) block Cheddarproduction lines were put into production. One 290 kg block replacedsixteen 18 kg blocks. The 290 kg block system reduced labor and handlingcosts, on-the-job lifting injuries, intermediate packaging costs, andtrim loss when blocks were converted to the exact weight pieces neededfor retail marketing.

However, although the handling of 290 kg blocks of cheese with forkliftswas efficient and easy, the cooling of the cheese in these large blocksimmediately after manufacture was more difficult. Thus, as the 290 kgblock systems became common in the industry, it became apparent that thecheese within the 290 kg blocks had variations in both composition andcheese quality. For example, in 1988, Reinbold et al. (J. Dairy Sci. 71:1499-1506) observed that after 7 days of cooling a 290 kg block ofcheese, moisture had traveled from areas of high to low temperature.Reinbold et al. also observed that after 24 hours of cooling, the curdhad not completely fused and was still porous.

Barbano et al. conducted systemic studies on 290 kg blocks of cheese andobserved that a moisture gradient of about 5% existed from the inside tothe outside of the cheese block. J. AOAC Intl. 84: 613-19 (2001). Thusthe center of 290 kg blocks of cheese was significantly drier than theoutside. Moisture was apparently wicking from the interior to theexterior during cooling of the cheese blocks, leading to irregularitiesand non-uniformities in cheese composition and quality. Smaller portionsof cheese cut for retail sale from these 290 kg blocks were sometimestoo wet, or too dry, depending upon what part of the block the retailportion was taken.

Hence, a problem exists in the cheese industry that threatens toundermine the efficient 290 kg block process routinely used for makingcheese.

SUMMARY OF THE INVENTION

The present invention provides a new approach to making cheese thatavoids the wicking, drying and moisture retention problems of existingprocedures. The present methods provide a uniformly moist block ofcheese with uniform composition and quality. The cheeses produced by themethods of the invention have excellent flavor, melt very well and canbe produced to retain more moisture than existing cheeses. The methodsof the invention are simple, and require less rennet and less salt thanexisting procedures.

The methods of the invention relate to controlling the pH of the cheesemaking process to optimize the partitioning of minerals and proteinsbetween curd and whey, and between the matrix and water phase withincurd particles.

Thus, the present invention involves a method for reducing watermigration in cheese that includes reducing the pH of pasteurized milkused for making the cheese to a pH of about 5.6 to about 6.2, beforeadding cheese-making starter cultures. The milk can be warmed to atemperature of about 85° F. to about 100° F. after the pH is adjustedand starter bacterial cultures can then be added to ripen and begin thecheese-making process. In some embodiments, the milk is acidified to apH of about pH 5.80 to about 5.85 when the milk is at a temperature ofabout 88° F. to about 95° F.

Milk typically has a pH of about 6.6 to about 6.7. Lowering the pH ofmilk helps the cheese making process and improves the cheese product ina variety of ways. For example, instead of being tightly bound toprotein, calcium tends to migrate into the soluble phase and becomesavailable to rennet, an enzyme required in a later stage of the cheesemaking process. Moreover, bacterial cultures used to initiate the cheesemaking process actually grow better under low oxygen conditions, and useof carbon dioxide to acidify the milk tends to drive some of the oxygenout of solution. Such low oxygen and high carbon dioxide levels optimizegrowth of cheese-making bacteria and inhibit growth of undesirablemicroorganisms that might otherwise contaminate the cheese-makingprocess. Acidification is also believed to move proteins such as caseininto the water phase. An increased protein content in the soluble phasehelps to hold water so that the cheese has a higher, more uniformmoisture content. Such a uniform increased moisture content helps thecheese to resist drying, promotes a longer shelf life and reduces cheesewaste and manufacturing costs. A higher protein content in the solublephase also helps the cheese to retain salt, not only reducing the amountof salt needed but also reducing salt run-off and the need to safelydispose of salt waste.

Thus, an improved cheese product is produced using the methods of theinvention. The improved cheese of the invention is uniformly moist,melts smoothly, has excellent flavor, has somewhat less fat (e.g. 5% to10% less fat) than cheese made without acidification, and has morecalcium and casein in a soluble phase of the cheese than does a cheesemade without acidification.

DESCRIPTION OF THE FIGURES

FIG. 1 graphically illustrates cheese pH versus time (minutes) duringcheese-making for cheeses made from control milk (◯) and milk to whichCO₂ has been added (□).

FIG. 2 shows cheese pH as a function of position within slab (n=3),position 1=bottom 2.54 cm of cheese slab and position 7=top 2.54 cm ofcheese slab. Control (⋄), average pH value over 3 weeks and CO₂-treated(◯), average pH value over 3 weeks.

FIG. 3 shows cheese moisture as a function of position within the cheeseslab (n=3), position 1=bottom 2.54 cm of cheese slab and position 7=top2.54 cm of cheese slab. Control (□), average moisture over 3 weeks andCO₂-treated (◯), average moisture over 3 weeks.

FIG. 4 graphically illustrates the mean (n=3) temporal pH pattern ofcontrol (squares) and CO₂-treated (circles) cultures during cheesemaking. Samples at times 0 and 45 min were milk, samples from 100 to 140min were whey, and samples after 140 min were curd.

FIG. 5 graphically illustrates the mean (n=3) CO₂ content of control andCO₂-treatment cheeses during 6 months of aging.

FIG. 6 graphically illustrates the mean (n=3) pH of the control andCO₂-treated cheeses during 6 months of aging.

FIG. 7 graphically illustrates the mean (n=3) titratable acidity of thecontrol and CO₂-treated cheeses during 6 months of aging.

FIG. 8 graphically illustrates the mean (n=3) soluble nitrogen as apercentage of total nitrogen (SNPTN) of the control and CO₂-treatedcheeses during 6 months of aging. Open symbols indicate pH 4.6 SNPTN andclosed symbols indicate 12% TCA SNPTN.

FIG. 9 graphically illustrates the mean (n=3) ratios of α_(s)-casein topara-κ-casein (open symbols) and β-casein to para-κ-casein (closedsymbols) in cheeses during 6 months of aging. Square symbols are usedfor control cheeses and circular symbols are used for CO₂-treatedcheeses.

FIG. 10 shows the proteins in expressible serum (ES) (25° C.) of Cheddarcheese, immediately after overnight pressing (about 16 h), separated bySDS-PAGE. Lanes 1 to 3 contain expressible serum of control cheeses fromthree cheese makings. Lane 5 is a whole milk reference sample. Lanes 7to 9 contain expressible serum from CO₂-treated cheese from three cheesemakings. Protein bands are identified on the gel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for making a flavorful,uniformly moist cheese that includes acidifying the milk used for cheesemaking just before the cheese making procedure is initiated. In general,a cheese making process involves milk pasteurization, warming the milkto a temperature of about 85° F. to about 105° F., incubating thewarmed, pasteurized milk with starter bacterial cultures to ripen themilk, adding rennet to coagulate the ripened milk, cutting the coagulateinto curd, healing the curd by stirring the curd/whey, raising thetemperature of the curd/whey suspension to about 90° F. to about 105°F., separating the curd from the whey, salting the curd, pressing thecurd into blocks and aging the blocks of cheese as needed. Theimprovement provided by the invention involves acidifying the milk to apH of about 5.6 to about 6.2, after pasteurizing the milk and beforeadding cheese making starter cultures. In some embodiments, the pH isadjusted to achieve a pH of about 5.8 to about 6.1 when the milktemperature is about 85° F. to about 105° F. Cheeses made by the presentmethods are uniformly moist, melt readily, have somewhat less fat, havemore calcium and have an excellent flavor. Thus, this improvement helpseliminate waste and improves the uniformity, composition and moisturecontent of the cheese product.

Acidification

Any acidifying agent can be used including carbon dioxide, vinegar,citric acid, lactic acid and the like. However, while the inventioncontemplates any acidifying agent, in some embodiments, carbon dioxideis preferred. Carbon dioxide has certain advantages, including the factthat carbon dioxide has essentially no flavor, carbon dioxide can act asan anti-microbial agent and carbon dioxide temporarily modulates the pHof cheese components during key cheese-making steps and then dissipatesover time as the carbon dioxide outgases. Hence, carbon dioxide acts asa processing aide and the majority of the carbon dioxide dissipates anddoes not form a substantial proportion of the final product.

The pH of the milk should be adjusted after pasteurization and aftercooling the milk from the pasteurization process. This is done to avoidany coagulation that may occur as a result of the combination of heatingand acidifying the milk. When the milk is acidified at cooltemperatures, no significant amount of coagulation should occur. Thus, acool temperature is used for acidification. Milk is usually pasteurizedby heating at 72° C. (161° F. to 162° F.) for 15 seconds to destroypotentially harmful bacteria. Milk is then typically cooled to around30° C. (86° F.). However, for the acidification procedures of theinvention, the milk should have a temperature of no greater than about10° C. (50° F.) before acidification is performed. In some embodimentsthe temperature is kept below about 7° C. (44° F. to 45° F.) beforeacidification is performed. In other embodiments, the temperature iskept below about 4° C. (39° F. to 40° F.) before acidification isperformed.

The pH of the milk should be reduced from the normal milk pH of about6.6 to 6.7, to a pH of about of about 5.6 to about 6.2, afterpasteurization and before adding cheese making starter cultures. In someembodiments, the pH of the milk can be reduced to an initial pH of about5.7 to about 6.1, or a pH of about 5.85 to about 6.05, or a pH of about5.9 to about 6.0 before addition of cheese-making cultures.

The pH can vary somewhat with temperature. Because the milk will beincubated with the starter bacterial culture at about 85° F. to about105° F., or at a temperature of about 90° F. to 100° F., or at atemperature of about 88° F. to about 95° F., the pH should be measured,adjusted and/or calculated at this temperature. Hence, an initial pH ofabout 5.8 to about 6.2, or a pH of about 5.9 to about 6.0 at about 85°F. to about 105° F. is desired. In some embodiments, the temperature isabout 90° F. to about 100° F. and the pH is about 5.9 to about 6.0.

When carbon dioxide is used to reduce the pH of milk, approximately 1000ppm to about 2000 ppm carbon dioxide are used. In some embodiments,approximately 1300 ppm to about 1900 ppm carbon dioxide are used, orapproximately 1400 ppm to about 1800 ppm carbon dioxide are used toachieve the desired pH.

Starter Cultures

Starter bacterial cultures are used to ripen and begin the cheese makingprocess. The starter cultures contain lactic acid producing bacteria isto help sour the milk and to convert lactose into lactic acid. Thishelps in the coagulation process. In addition, the starter cultures alsohave a beneficial effect on the eventual quality, taste and consistencyof the cheese.

Starter cultures typically include live cultures of lactic acid bacteriasuch as, for example, Streptococcus thermophilus and Lactococcuscremoris bacteria. These bacteria naturally produce lactic acid andnaturally lower the pH of the ripening milk used during cheese making.The methods of the invention accelerate the pH lowering process andfacilitate bacterial action. Use of carbon dioxide as the acidifyingagent minimizes oxygen content in the milk culture, further enhancingbacterial action.

Any available cheese making starter cultures can be used with themethods of the invention. For example, commercially available cheesemaking starter cultures such as 911 DVS pellets (Chr. Hansen Inc.,Milwaukee, Wis.) can be employed. Ripening by starter cultures can bedone for about 30 minutes to about 90 minutes at a temperature of about85° F. to about 100° F. During this process the pH will typically remainat about 5.6 to about 6.2.

Coagulation

The ripened milk is coagulated by the addition of rennet. The activeingredient of rennet is the enzyme, chymosin (also known as rennin). Anyavailable rennet can be used in the invention. One source of rennet isthe stomach of slaughtered newly-born calves. Vegetarian cheeses aremanufactured using rennet from either fungal or bacterial sources.Advances in genetic engineering processes have made recombinantlyproduced chymosin available. Any of these rennet types can be employedin the invention. For example, rennet can be obtained commerciallyChymax Extra from Chr. Hansen Inc. (Milwaukee, Wis.).

The amount of rennet employed can be reduced when employing the methodsof the invention. In general, when the pre-acidification methods of theinvention are used, the amount of rennet employed can be about one-thirdto about two-thirds of the rennet used for making cheese withoutpre-acidification. In some embodiments, the amount of rennet employedafter pre-acidification is about one-half that used when noacidification is performed. Hence, the methods of the invention can beless expensive than currently available methods because lower amounts ofrennin can be used.

The exact amount of rennet employed depends upon the activity of theenzyme. When using Chymax Extra, about 0.05 to about 0.2 milliliters perkilogram of milk can be used; in other embodiments about 0.1 mL/kg ofmilk is used. The temperature employed during coagulation with rennetcan vary. In some embodiments, the ripened milk is incubated at atemperature somewhere between room temperature and body temperature, forexample, at about 20° C. (68° F.) to about 37° C. (98.6° F.), or atabout 29° C. (84.2° F.) to about 33° C. (91.4° F.).

The coagulation time can also vary. In general, the milk is allowed tocoagulate for about 10 minutes to about 40 minutes, or about 15 minutesto about 30 minutes, or about 20 minutes to about 25 minutes.

Curd Production

After treatment with rennet, the coagulum is cut to form curds ofdesired size. The curds and whey are not stirred for a short while, forexample, about 2 min to about 10 min to allow the curds to heal. In someembodiments, the curds and whey are not stirred for about 5 min to healthe curds. After healing, the curds and whey are stirred gently withoutadded heat for 10 min.

Note that the curds will float when carbon dioxide is used as theacidifying agent. Hence, cheese vats may need to be adapted to allow forcurds that float rather than sink.

After a brief resting period, the temperature is increased a few degreesover a 15 minute time period, to a temperature of about 35° C. (95° F.)to about 40-41° C. (105° F.), or about 37.8° C. (100° F.). At the end ofthis heating process, some whey can be removed, cooled to about 4-5° C.(40° F.) and saved for later use. The remaining curds and whey arecontinuously stirred at a temperature of about 35° C. (95° F.) to about40° C. to 41° C. (105° F.), or at about 37.8° C. (100° F.) until thecurd pH reaches about 5.2 to about 6.2, or about 5.4 to about 6.0.

When the curd pH is about 5.2 to about 6.2 (or in some embodiments,about 5.4 to about 5.8), cool the curd and whey to a temperature ofabout 83° F. or lower by addition of the cold whey that was previouslyremoved at the end of the heating process. The addition of this coldwhey (or cold UF permeate) helps to cool the curd/whey suspension.Cooling the curd in liquid (e.g. whey) until the target pH of about 5.2to 6.2 is reached, causes the curd to absorb moisture and increasesfinal cheese moisture. Addition of cooled whey is preferred overaddition of water because whey is more flavorful. Also, this is a gooduse for the whey and reduces waste.

This combination of curd pH and temperature of the whey is an importantstep in moisture control and pH control. When the pH of the cooledwhey/curd suspension is appropriate (about pH 5.2 to pH 6.0, or about5.6), the whey is drained off.

The curds can then be salted. Three applications of salt can be madewith mixing between applications. The curd is drained and salted at a pHand temperature low enough to achieve the desired final moisture and pH.The combination of low temperature and salt slows down the culture sothat the pH does not go too low. Proper combinations of lower pH's andlower temperature's can be used to achieve a higher cheese moisturewithout having the final pH of the cheese go too low. Note that lesssalt can be employed in the cheeses of the invention because the salt isretained better by the curds produced according to the methods of theinvention. After salting, the curds can then be pressed into blocks orotherwise processed for aging, storage and/or sale.

In many embodiments, the cheese can be packaged for immediate use orfrozen for later use. If the cheese is frozen, the desired final pH ofthe cheese after freezing and thawing is in the range of about 5.1 toabout 5.4, with a final percent moisture of greater than 53%. Dependingon the combination of pH and temperature used prior to salting, cheesemoistures of over 60% can be achieved. Such high moisture cheeses areuseful because they remain moist for longer periods of time (therebyavoiding drying), they tend to be lower fat (more water, less fat) andthey are less expensive to produce.

If a shredded cheese is desired, the block cheese can be cut or shreddedinto small particles. Most cheese-making procedures require an agingperiod before the cheese can be shredded. However, the present methodscan eliminate this aging step. If desired, a hydrophobic surface coatingcan be sprayed onto the particles to help separate the particles andmodify the melting characteristics of the shredded cheese. Shreddedcheeses made by the process of the invention can be used on top of avariety of frozen and non-frozen food products including pasta, chickendishes, veal dishes, vegetables and the like. Because the cheeses of theinvention are so moist, there is no need to add starch or another agentthat improves moisture retention.

Cheeses produced by the method of the invention can have a fat on drybasis (FDB) value of about 40% with a moisture content of 53 to 54%,salt content 1.5 to 1.7, pH of about 5.2. Such cheeses can be packagedand sold as cheese blocks, or as shredded cheese. The cheeses of theinvention can also be used in a variety of food products.

Benefits of Using Carbon Dioxide

Use of carbon dioxide has several beneficial effects upon thecheese-making process and upon the ultimate cheese product. For example,carbon dioxide causes a shift in equilibrium of calcium in the milkmoving some of the calcium that is bound to casein into the whey. Thisshift in calcium enhances the milk coagulation action of rennet andpermits less rennet to be used (about 50% less). The removal of boundcalcium from casein by carbon dioxide early in the cheese-making processis also important for achieving excellent meltability of the cheesewithout aging the blocks at refrigeration temperature for several daysbefore shredding (and freezing, if desired).

The removal of bound calcium from casein early in the process due theaction of CO₂ causes changes the casein matrix structure of the curdparticles. When this curd is salted and cooled, more casein moves fromthe matrix and dissolves in the water phase of the cheese than when CO₂is not used. The casein that has moved into the water phase binds waterand allows the cheese to achieve a higher moisture content while stillholding the water (without starch) during melting or baking. Thus, nostarch or other material need be added to cheeses of the invention toimprove the meltability and water retention of the cheese.

Moreover, curd produced by the present methods binds water takes up saltmore efficiently during the salting process. This reduces salt loss,salt waste and the cost of making cheese.

Summary of a Preferred Cheese-Making Procedure

-   -   1. Pasteurize the skim (or standardized milk) milk, cool the        milk and inject CO₂ into milk. The injection of carbon dioxide        is generally performed at a milk temperature of 40° F., but some        variation in temperature is permitted    -   2. The milk is heated to 93 to 95° F. The level of CO₂ in the        milk needs to be sufficient to produce a milk pH of about 5.90        to 6.0 at a temperature 90 to 100° F.    -   3. Add starter culture (for example, mixed cultures of        Streptococcus thermophilis and Lactococcus cremoris) and ripen        45 minutes.    -   4. At the end of ripening, add rennet. The amount of rennet used        can be about 50% of that used when no CO₂ is used in the cheese        making. Let the milk coagulate (about 20 to 25 minutes) and then        cut it.    -   5. After cutting, allow a 5 min curd heal and then stir gently        and heat from about 95° F. to 101° F. in 15 min. If cheese vats        are used, they may be adapted to allow for curd that floats        instead of sinks.    -   6. Remove some hot whey at the end of heating and cool this whey        to 40° F. Continue stirring the curd and the remaining whey at        95° F. to 101° F. until the curd pH reaches 5.8.    -   7. At a curd pH of 5.4-5.8, cool the curd from 95° F. to 100° F.        by addition of cold whey to reach a temperature of about 83° F.        or lower. The combination of curd pH and temperature are used to        control the final pH and moisture of the cheese.    -   8. At a curd pH of 5.6 or less, drain the whey. Add salt to the        curd in 3 applications with about 5 minutes of mixing between        applications. The approximate time from rennet addition to        salting is about 110 to about 1600 minutes.    -   9. Immediately put the salted curd into hoops and press for 1        hour at 40 psi.    -   10. After 1 hour remove the block cheese from the hoop, place it        into a plastic bag and vacuum seal it.    -   11. Place the block (packaged in plastic) into ice water to        rapidly cool it. The block cheese is ready to be shredded and/or        quick frozen the next day.    -   12. The cheese can be thawed and packaged directly with or        without a hydrophobic surface spray to enhance meltability.

When the procedures of the invention are used, the aging of cheese caneffectively be eliminated. Moreover, cheese made by the procedures ofthe invention can be frozen and thawed before use without separation offat from water, loss of moisture or adverse effects upon the texture,taste and melting properties of the cheese. If the cheese will be frozenimmediately after it is made, the desired final pH of the cheese afterfreezing and thawing is in the range from 5.1 to 5.4, the finalmoisture >53%, and the final pH <5.4. The combination of pH andtemperature used prior to salting can influence the final pH andmoisture content. Final cheese moistures of over 60% can be achievedusing the methods of the invention.

Types of Cheese

All types of cheese can be made by the present methods. For example,American, Cheddar, Monterey Jack, mozzarella, Muenster, Swiss, and thelike can be made by the present methods. In some embodiment, all typesof Cheddar cheese and mozzarella cheeses are made by the presentmethods.

Moreover, low-fat cheeses can be made by the methods of the invention.As illustrated herein, use of the pre-acidification methods of theinvention leads to a cheese product that has a lower fat content than dosimilar methods that do not employ pre-acidification. Thus, for example,the fat content of the cheeses produced according to the invention canhave about 2% to about 50% less fat, or about 3% to about 30% less fat,or about 4% to about 20% less fat, or about 5% to about 10% less fatthan cheeses made without pre-acidification. Fat was reduced mostlyduring draining of whey.

Cheese Composition, Flavor and Texture

The composition of the cheeses produced by the methods of the inventionis improved in several respects. First, the cheese is uniformly moist.Second, the moisture content of the present cheeses is somewhatincreased, for example, from about 50% up to about 60%. In someembodiments, the cheeses of the invention have a moisture content ofabout 53% to about 54%. Such an increase in moisture prevents the cheesedrying out and improves the economics of cheese production. Third, thecheese does not bleed moisture. It is believed that the moisture in thecheese is retained by the higher content of protein in the soluble phaseof the cheese.

The flavor of cheeses produced by the methods of the invention is notadversely affected by the pre-acidification procedure and is generallyimproved by the increased moisture, improved salt retention and improvedmelting characteristics of the cheese. The intensity of cheese flavorresults primarily from the action of enzymes, starter organisms, andnonstarter culture bacteria on intact casein and the degradationproducts of casein and amino acids. Aston and Creamer (1986) havereported that a sub-fraction of the water-soluble fraction containingmost of the salt, free methionine, and free leucine contributed the mostto the flavor of the water soluble fraction. By retaining andstabilizing the moisture naturally produced by the cheese, the flavor ofthe cheeses produced by the present methods is improved.

The texture of cheeses is typically due to the proteolytic breakdown ofthe casein matrix and possibly to changes in casein-water-calciuminteractions as a function of aging. For example, Cheddar cheese texturestarts out rubbery and corky, but rapidly changes to a softer moresmooth texture as proteolysis continues during aging.

The methods of the invention improve the texture of cheese in severalrespects. Addition of an acidifying agent shifts the equilibrium ofcalcium from being bound to casein to being in solution within the whey.The removal of bound calcium from casein also shifts theinsoluble-soluble casein equilibrium towards solubility. Thus the matrixstructure of casein particles changes. This change in the casein matriximproves the melting characteristics of cheese without the need forextensive aging.

Thus texture of cheese made by the procedures of the invention ismoister, smoother and dissipates even more quickly in the mouth thancheese made without use of acidifying agents.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLE 1 Comparison of Procedures for Controlling Moisture Migration inCheese

In this Example, three approaches to reducing moisture migration in 290kg blocks were evaluated: (1) addition of hydrocolloids (e.g., starch,cellulose) to milk during cheese making, (2) addition of denatured wheyproteins (e.g., Simplesse®) to milk during cheese making, and (3)addition of CO₂ to milk to shift casein monomers and calcium from themicelles into the water phase of the milk, prior to rennet addition, tobind water and reduce moisture mobility in the cheese. Some of thematerials in the first two approaches increased the moisture content ofthe cheese, but did not make a major reduction in moisture migrationduring cooling of the cheese. The addition of CO₂ to milk was veryeffective at reducing moisture migration in cheese during cooling andthe results of that work are reported below.

Materials and Methods

Milk Carbonation

The milk carbonation system was a countercurrent stainless steel tubularheat exchanger (internal diameter=0.5 cm) circulated with 0 to 1° C.water. The carbonation system consisted of four units. They weresequentially from inlet to exit: (1) a milk feed reservoir; (2) aperistaltic feed pump (Amicon LP-1 pump, Beverly, Mass. with aCole-Palmer Masterflex® 7015-81 pump head, Vernon Hills, Ill.) which fedthe milk (4° C.) into the carbonation system at a flow rate of 900 m/mm;(3) a CO₂ injection port, which was a stainless steel tube (internaldiameter=0.08 cm) inserted through a tee-fitting perpendicular to themilk flow; and (4) a holding section in which the milk was kept at 2 to3° C. Throughout the system, several temperature probes were insertedinline through tee-fittings to monitor milk temperature. CO₂ (beveragegrade) was injected inline into cold skim milk as it entered thecarbonation system. The CO₂ injection port was connected to a CO₂ tank[CO₂ line pressure=55 psi (380 Kpa)] and the flow rate of CO₂ (1110ml/min) was controlled with a flow meter. The CO₂ flow rate wasdetermined in a preliminary experiment to achieve a target concentrationof CO₂ in milk of approximately 3000 ppm. Carbonation of pasteurizedskim milk was done at 2 to 3° C.

Cheese Making Procedure

Reduced-fat Cheddar cheese was made by transferring either 215 kg of 4°C. pasteurized carbonated, or 215 kg of noncarbonated, skim milk to acheese vat (model 4MX; Kusel Equipment Co., Watertown, Wis.). The caseinto fat ratio was standardized by adding non-carbonated pasteurized heavycream (approximately 40% fat) to achieve a 50% fat reduction (comparedto full-fat Cheddar cheese) in the final cheese. A stirred-curdcheesemaking procedure was used, as previously described in Olabi andBarbano, J. Dairy Sci. 85: (2002).

The amount of chymosin and starter added per unit weight of milk was thesame for carbonated and noncarbonated milk and the time from chymosinaddition to cutting was 30 min. The coagulum was cut with 1.2 cm wirecheese knives and then allowed to heal for 5 min. Healing was followedby slow agitation for 10 min at 31° C. The temperature was increasedfrom 31 to 33° C. and from 33 to 37° C. in two 15 min intervals. Thecurd plus whey was stirred at 37° C. until a whey pH of 6.2 wasachieved. Because the pH of the whey produced from carbonated milk wasalready lower than 6.2 (mean of three cheese-making trials was 6.01),the whey was drained, as soon as the second phase of cooking (at 37° C.)ended.

At this point most of the whey was drained and the remaining curd-wheymixture was stirred at 37° C. until a curd pH of 5.8 was attained. Next,additional whey was removed and cold water (about 5° C.) was added untila curd temperature of 28° C. was achieved. Stirring was continued untila curd pH of 5.6 was achieved. Next, the mixture of whey plus water wasdrained from the curd, the curd was weighed, and salt was added (totalof 2.7% wt/wt) in three equal portions with a 5-min interval betweenapplications. The curd plus salt was mixed for about 1 min after eachsalt addition. The salted curd was put into an 18 kg stainless steelWilson hoop and pressed, using a hydraulic A-Frame press (Model AFVS,Kusel Equipment Co., Watertown, Wis.), at 10 psi (70 kPa) for 30 minfollowed by 60 psi (420 kPa) for 4.5 h at room temperature.

Sampling

Immediately after the 18 kg blocks of cheese (one bock for eachtreatment) were removed from the press they were cut and sampled in awalk-in climate control room at 27±0.5° C. The internal temperature ofthe blocks of cheese was approximately 25° C. Each 18 kg pressed blockwas cut in half at the middle and then three 17.78×7.62×2.54 cm (length,width, thickness) slabs of cheese were obtained from the center of eachblock. These slabs were immediately vacuum packaged (Multi Vac model160; Koch, Kansas City, Mo.) in 3-mil polyethylene bags (25.4×45.7 cm,standard barrier; Koch, Kansas City, Mo.). In addition, two slabs of28.6×14.0×2.54 cm (length, width, thickness) were cut from the 18 kgcheese block and vacuum packaged, as described above. The two slabs werecooled immediately and stored at 4° C. One slab was used for the cheesecomposition analysis and the other slab was used for the expressibleserum analysis.

After the 17.78×7.62×2.54 cm cheese slabs were vacuum packaged, each bagwas marked with horizontal lines to identify each 2.54 cm position. Thedifferent positions were numbered from the bottom (1) to the top (7).The slabs were attached to the suspension wires on a temperaturegradient apparatus designed to cause moisture migration upwards fromposition 1 to position 7. The apparatus consisted of a water bath and arotating cylinder designed to gradually raise vacuum packaged slabs ofcheese out of the 27° C. water into the 3° C. air over a period of 36 h.

The 3° C. slabs of cheese were removed from the apparatus after 36 h andcut with a knife into 2.54 cm pieces by position. The cheese from eachposition was ground in a blender (model 31BL92; Waring, New Hartford,Conn.) and placed into two 50 ml plastic snap-lid vials (leaving no headspace) and held at 4° C. after blending. The pH of cheese from eachposition within each slab was measured at 23° C. as a single measurementwithin 2.25 h of grinding and the moisture content of the cheese withineach position was measured in triplicate within 24 h after sampling.

Chemical Analyses

Titratable acidity of the milk and whey was determined as described inMarshall (1992) Chemical and physical methods. Pages 433-529 [methodnumber 15.3.A] in Standard Methods for the Examination of Dairy Products(16^(th) ed.) American Public Health Association, Washington, D.C. ThepH of the milk, whey and cheese was determined using pH model HA405,Mettler Toledo electrode, Columbus, Ohio and Accumet pH meter, model915; Fisher Scientific, Fair Lawn, N.J.) during cheese manufacturing.The electrode was immersed in 3 M KCL storage solution at 38° C. betweenpH measurements to improve response speed and stability. Referencesolutions (Fisher Scientific, Fair Lawn, N.J.) for pH 4 (SB 101-500) andpH 7 (SB 107-500) were used at 38° C. for calibration of the pH meter.The actual pH of the reference buffers was calculated for 38° C. basedon the temperature coefficients recommended by the buffer manufacturer.

After cheese manufacture, the fat content of milk was determined by theBabcock method for milk (Association of Official Analytical Chemists,Methods of Analysis (17^(th) ed. 2000); method number 33.2.27; 989.04)and whey by skim milk Babcock test [(Marshall, 1992); method number15.8.B] modified for use at 48° C., instead of 58° C. for tempering andreading the fat columns (Lynch et al., JAOACI. 80. 845-859 (1997)).Total nitrogen (TN) for the milk and whey was determined by Kjeldahl((Association of Official Analytical Chemists, 2000); method number33.2.11; 991.20). Non-protein nitrogen in milk and whey was determinedby Kjeldahl ((Association of Official Analytical Chemists, 2000); methodnumber 33.2.12; 991.21). Noncasein nitrogen (NCN) was determined byKjeldahl ((Association of Official Analytical Chemists, 2000); methodnumber 33.2.64; 998.05). The casein content was calculated as TN minusNCN multiplied by 6.38. Calcium was determined by an atomic absorptionspectroscopy procedure of Brooks et al. (Atomic absorption Newsletter9(4): 93-94 (1970)), as modified by Metzger et al. (J. Dairy Sci.83:648-58 (2000)). The ppm CO₂ content of the milk was determined usingan infrared gas analysis method described by Ma et al. J. Dairy Sci. 84:1959-68 (2001). All analyses were performed in duplicate.

To prepare cheese for analysis of moisture, fat, protein, salt, calcium,and pH, the cheese from the 28.6×14.0×2.54 cm slab was ground in ablender (model 31BL92; Waring, New Hartford, Conn.) to a particle sizeof 2 to 3 mm. The ground cheese particles were packed into 50 ml plasticsnap-lid vials (leaving no head space) and immediately placed in a 4° C.refrigerator. Fat content was determined by the Babcock method (Marshall(1992) Chemical and physical methods. Pages 433-529 in Standard Methodsfor the Examination of Dairy Products (16^(th) ed.). American PublicHealth Association, Washington, D.C.; method number 15.8.A). Todetermine fat content 9 g of cheese was used with 12 ml of distilledwater at 100° C. Total nitrogen was determined by Kjeldahl using a 1 gsample size. See, Association of Official Analytical Chemists (2000)Official Methods of Analysis. 17^(th) ed. AOAC, Gaithersburg, Md.(method number 33.2.11; 991.20). Salt content was determined by theVolhard test ((Marshall, 1992); method number 15.5.B.); and moisture wasdetermined gravimetrically, by drying 2 g of cheese at 100° C. in aforced air oven (model OV-490A-2; Blue M, Blue Island, Ill.) for 24h)(Association of Official Analytical Chemists, 2000); method number33.2.44; 990.20). Calcium was determined by an atomic absorptionspectroscopy procedure of Brooks et al. (Atomic Absorption Newsletter9(4): 93-94 (1970) and as modified by Metzger et al. (J. Dairy Sci. 83:648-58 (2000). Cheese pH was measured at 23° C. The analyses were donein duplicate for pH, salt, and calcium, in triplicate for fat and totalnitrogen, and in quadruplicate for moisture. The amount (g/100 g ofcheese) of expressible serum was determined at d 2 of refrigeratedstorage, as described by Guo and Kindstedt (J. Dairy Sci. 78:2099-2107(1995)) with one modification. The cheese serum was removed bycentrifugation at 25,000×g (maximum force) instead of 12,500 (maximumforce).

Experimental Design and Statistical Analysis

Two vats of reduced-fat Cheddar cheese, one from non-carbonated and onefrom a carbonated portion of the same milk, were made side by side ineach of 3 wk. One 18 kg block of reduced fat Cheddar cheese was producedfor each treatment in each of the three weeks. On each week, threecheese slabs for each treatment were removed from each 18 kg block, asdescribed by Olabi and Barbano, J. Dairy Sci. 85: (2002). The cheeseslabs were put on the apparatus using a temperature gradient designed tomove moisture upward (id.). In the ANOVA model, treatment was a categoryvariable and position was a continuous variable, while cheese makingweek (i.e., batch of milk) was blocked as a fixed effect. The positionvariable was transformed to make the data set orthogonal. Therefore,positions 1, 2, 3, 4, 5, 6 and 7 were coded as −3, −2, −1, 0, +1, +2,and +3, respectively, as the input data for the position variable in theANOVA. The interaction term between treatment and cheese making week andposition was used as the error term for the main effects. The PROC GLMprocedure of SAS was used for all data analyses (SAS version 8.02,1999-2001).

Results

Milk Composition and CO₂ Content

No significant difference (P>0.05) in fat, protein, casein, and NPNcontent of the standardized milk with and without added CO₂ was detected(Table 1).

TABLE 1 Mean (n = 3) milk, whey, and cheese composition for control andCO₂ treatments. Treatment Sample type CONTROL CO₂ LSD Milk Fat, %  1.23  1.23 NS Protein, %  3.06   3.01 NS Casein, %  2.23   2.20 NS NPN¹, % 0.20   0.21 NS CO₂ at 4° C.², ppm 139^(B) 3175^(a) 800 CO₂ at 31° C.³,ppm 110^(B) 1721^(a) 523 pH at 31° C.  6.63^(A)   6.01^(b) 0.05 TA⁴ at31° C.  0.16^(B)   0.42^(a) 0.09 Whey Fat, %  0.05   0.06 NS Protein, % 0.80   0.79 NS NPN¹, %  0.27   0.26 NS Cheese Moisture, %  47.72^(b) 53.48^(a) 3.01 Fat, %  15.17^(a)  13.61^(b) 0.64 FDB, %  29.00  29.26NS Protein, %  30.59^(a)  26.64^(b) 1.69 PDB, %  58.51^(a)  57.27^(b)0.78 M/P⁵  1.56^(b)   2.01^(a) 0.22 Salt, %  2.04^(b)   2.40^(a) 0.28S/M⁶, %  4.27   4.50 NS pH  5.08   5.09 NS ^(a,b)means within row nothaving a common superscript differ (p < 0.05). ¹NPN expressed asnonprotein nitrogen times 6.38. ²CO₂ at 4° C. = CO₂ content ofpasteurized carbonated skim milk. ³CO₂ at 31° C. = CO₂ content of vatmilk collected just before the addition of starter culture. ⁴TA =titratable acidity. ⁵M/P = moisture to protein ratio. ⁶S/M = saltconcentration in moisture.

The titratable acidity of the milk with added CO₂ was very high(Table 1) and reflects the degree of interaction of CO₂ with water toform carbonic acid in the milk. The system for addition carbon dioxideto skim milk used in this study increased (P<0.05) the level of CO₂ inthe 4° C. skim milk prior to cheese making and achieved our target ofapproximately 3000 ppm of CO₂ (Table 1). After addition of cream, thatdid not contain CO₂, to the skim milk, the mixture was stirred andheated from 4° C. in an open cheese vat to 31° C. prior to cheesemaking. During this process some CO₂ was lost from the milk. The CO₂content of the standardized milk at the point of starter cultureaddition at 31° C. had decreased from about 3000 ppm to about 1721 ppm(Table 1).

Addition of CO₂ to milk caused a decrease in milk pH from 6.63 to 6.01at 38° C. (Table 1), as was expected from the results of Ma et al., J.Dairy Sci. 84:1959-1968 (2001). Ma et al. (2001) also reported acorresponding decrease in milk freezing point as the concentration ofCO₂ in milk increased and pH decreased. The decrease in freezing pointreported by Ma et al. (2001) is caused by the combined effects of thedissociation products of carbonic acid and the shift in equilibrium frombound calcium phosphate in the casein micelles to soluble calciumphosphate in the serum phase of milk. De La Fuente et al. (J. Food Prot.61:66-72 (1998)) reported that addition of HCl, lactic acid, and CO₂ tomilk to produce a reduction of milk pH to 6.1, all produced an increasein the proportion of soluble calcium in cow's milk and a similar effectwas reported for sheep and goat milks. The increase in soluble calciumcontent, caused by CO₂ addition to milk, would be expected to influencemilk coagulation properties.

Lowering the pH of milk also has an effect on micelle structure. As thepH of milk is lowered, a considerable portion of the micellar caseinsolubilizes. Dalgleish and Law, J. Dairy Res. 55:529-538 (1988); Singhet al., J. Dairy Sci. 79:1340-1346 (1996); van Hooydonk et al., Neth.Milk Dairy J. 40:281-296. (1986). In the case of the milk containing CO₂prior to cheesemaking, this means that a higher level of soluble caseinshould have been present in the serum phase of milk prior to rennetaddition. It was not clear if these nonmicellar caseins would be trappedin the rennet coagulation or if they will be lost in the whey.

Cheesemaking

The curd was visibly firmer at 30 min and started to coagulate soonerafter rennet addition for the milk with added CO₂ than for milk withoutCO₂. Okigbo et al. ((1985) J. Dairy Sci. 68:3135-3142), found thatreducing milk pH prior to rennet addition increased the speed of milkcoagulation with rennet. Other workers have reported decreasedcoagulation time. (Calvo et al. (1993) J. Food Prot. 56: 1073-1076; Dela Fuente, et al. (1998) J. Food Prot. 61:66-72). Further work indicatesthat addition of CO₂ may reduce the amount of rennet used in cheesemaking (McCarney, et al. (1995) Milchwissenschaft 50:670-674; Montillaet al. (1995) Z. Lebensm Unters Forsch. 200.289-292; St-Gelais et al.(1997) Milchwissenschaft 52: (11) 614-618).

The decision of when to drain the whey in our cheese making was based onthe pH of the whey at the end of the cooking step. The milk with addedCO₂ had a lower whey pH than the target whey pH than generally used fordraining in the inventor's cheesemaking procedure and therefore the wheywas drained immediately after the final cook temperature of 37° C. wasachieved. As a result, the time from starter addition to whey drainingwas shorter (P<0.05) for milk with added CO₂, but the time from wheydraining to the time when a curd pH of 5.6 was achieved (i.e., saltaddition) was longer (P<0.05) for the milk with added CO₂. This produceda total make time from starter additional to salt addition that was notdifferent (P>0.05) for milk with or without added CO₂ (Table 2).St-Gelais et al. (1997) reported a 30 min reduction in total cheesemaking time due to the preacidification of milk to pH 6.55 with eitherCO₂ or lactic acid. This effect was not observed in the experimentsreported herein.

TABLE 2 Mean make times (n = 3), (min.) for control and CO₂ treatments.Treatment Cheesemaking step CONTROL CO₂ LSD Starter addition to wheydraining 164^(a) 128^(b)  2.9 Whey draining to salt addition  83^(b)129^(a) 10.8 Starter addition to salt addition 247 256 NS ^(a,b)meanswithin column not having a common superscript differ (p < 0.05).

The profile of pH change with time during cheese making for milk withadded CO₂ was quite different than for milk without added CO₂, as shownin FIG. 2. Milk pH starts out low (about 6.0) and decreased very slowlyfor the first 200 min of the cheese making process for the milkcontaining approximately 1700 ppm of CO₂ (FIG. 2). During this time,lactic acid was being produced by the starter culture and CO₂ was beinglost from the curd plus whey, with the net effect being a small and slowdecrease in pH from about 6.0 to 5.8 over a period of about 200 min.This difference in pH profile during the cheese making process had amajor impact on the calcium content of the whey and the cheese.

Whey and Cheese Composition

There were no significant differences in the fat, protein, or NPNcontents of the whey from cheese making between control and milk withCO₂ (Table 1). If the addition of CO₂ did cause casein monomers to moveout of the casein micelles along with some calcium phosphate prior torennet addition, the presence of these caseins outside the micelles didnot result in an increase in protein content of the whey.

The control cheese had similar composition to reduced-fat Cheddar cheeseproduced in other research studies (Johnson et al. (2001) J. Dairy Sci.84: 1027-1033; Fenelon et al. (1999) J. Dairy Sci. 82:10-22; Chen et al.(1998) J. Dairy Sci. 81:2791-2797; Metzger and Mistry (1995) J. DairySci. 78:1883-1895). The moisture content of the cheese made with CO₂added to the milk was almost 5% higher (P<0.05) than the reduced fatCheddar made from the same milk without CO₂. McCarney et al. ((1995)Milchwissenschaft 50:670-674), also reported that moisture content oftheir Cheddar cheese made from milk containing CO₂ was higher despitemodifications to the manufacturing process to reduce moisture levels.However, they did not provide any moisture data in their report, so itis not possible to determine the magnitude of the effect they observed.Johnson et al. ((2001) J. Dairy Sci. 84: 1027-1033) reported that afirmer coagulum at cutting favors higher moisture content. In our studythe coagulum at cutting for milk with added CO₂ was much firmer thanwithout added CO₂ and the final moisture content of the cheese washigher. As expected, the higher moisture content of the cheese in thisstudy caused the concentration of both fat and protein (on a wet basis)to be lower (P<0.05) due to dilution. The fat on dry basis (FDB) in thecheese was not influenced by the use of CO₂, but the protein on a drybasis (PDB) was slight lower (P<0.05) for cheese made from milk withadded CO₂. Further work with measurement of cheese yield would be neededto determine if the lower PDB is an indication of slightly higherprotein loss in salt whey during pressing. The higher moisture contentof the cheese produced with CO₂ added to the milk results in a muchhigher moisture to protein ratio and would tend to produce a softercheese (Table 1). The absolute concentration of salt in the cheese madefrom milk with added CO₂ was higher (P<0.05) than cheese made from milkwithout added CO₂, but no difference in the salt concentration in thewater phase of the cheese was detected (P>0.05). The final pH values ofthe cheeses were almost identical (Table 1).

As shown in Table 3, there was a major influence of the addition of CO₂to the milk on the calcium content of the whey and cheese.

TABLE 3 Mean (n = 3) milk, whey, and cheese calcium levels (g/100 g) forcontrol and CO₂ treatments. Treatment Sample type CONTROL CO₂ LSD Milk0.110 0.109 NS Whey 0.046^(b) 0.066^(a) 0.005 Cheese 0.789^(a) 0.508^(b)0.102 ¹Ca/P 2.58^(a) 1.90^(b) 0.251 ^(a,b)means within row not having acommon superscript differ (p < 0.05). ¹Ca/P = calcium as a percentage ofprotein in cheese.The calcium content (Table 3) of the whey was higher (P<0.05) and thecalcium content of the cheese was lower (P<0.05) from milk with addedCO₂. The increase in calcium content of the whey that was observed isconsistent with the shift in bound to soluble calcium in milk duringacidification as reported by Le Graet and Brulé ((1993) Lait 73:51-60).In another study, Metzger et al. ((2000) J. Dairy Sci. 83: 648-658)acidified milk to pH 6.0 prior to low fat Mozzarella cheese making withacetic and citric acid and reported higher concentrations of calcium inwhey and lower concentrations of calcium in cheese. Metzger et al.((2001) J. Dairy Sci. 84: 1348-1356) reported that reducing the pH ofmilk prior to cheese making reduced the hardness, initial apparentviscosity, and expressible serum of low fat Mozzarella cheese. While notexture measurements were done on the reduced fat Cheddar cheese made inthe current study, the higher moisture content and lower calcium contentfor the cheese made from milk with added CO₂ produced a cheese that wasvisibly softer than the cheese made without CO₂ added to the milk.

Expressible serum content of cheese can be used to reflect the status ofwater mobility within the structure of the cheese. The mobility of waterwithin the cheese structure could be very important in moisturemigration during cooling of 290 kg blocks of Cheddar cheese. Theexpressible serum content of the reduced fat Cheddar cheese produced inthe current study was measured on d 2 after cheese making and was foundto be much lower (P<0.05) in the cheese that was produced from milk withadded CO₂ (Table).

TABLE 4 Mean (n = 3) cheese expressible serum at day 2 (g/100 g cheese)for control and CO₂ treatments. Treatment Sample type CONTROL CO₂ LSDCheese 7.487^(a) 0.931^(b) 3.66 ^(a,b)means within row not having acommon superscript differ (p < 0.05).This low expressible serum content may have an impact on water mobilitywithin large blocks of Cheddar cheese during cooling. The resultsprovided herein are consistent with the results of Metzger et al.((2001) J. Dairy Sci. 84: 1348-1356), who reported that reduction inmilk pH prior to cheese making produced Mozzarella cheese with a loweramount of expressible serum. It is normal for expressible serum contentof cheese to decrease with time during refrigerated storage (Guo andKindstedt (1995) J. Dairy Sci. 78:2099-2107) and after about 2 wk ofstorage of Mozzarella cheese, the amount of expressible serum is nearzero. Guo et al. ((1997) J. Dairy Sci. 80:3092-3098) also reported thatthe amount of expressible serum in the structure of cheese immediatelyafter manufacture was reduced by the addition of salt. These changes inthe mobility of water can influence, or are at least related to,functional properties such as meltability of cheese. McMahon et al.((1999) J. Dairy Sci. 82:1361-1369) described water partitioning inMozzarella cheese and its relationship to cheese meltability. They foundthat the amount of chemically bound water in cheese remained constantduring refrigerated storage of cheese but that there was an increase inentrapped water and a decrease in expressible water with increasing ageof the cheese and that these changes were related to time dependentchanges in cheese functionality. Assuming that a similar behaviorhappens in Cheddar cheese, the amount of water bound to protein probablyremained constant in our cheese but the CO₂ treatment caused a largedecrease in the expressible water (i.e., easily movable) and an increasein the entrapped water within the structure of the reduced fat Cheddarcheese. This increase in entrapped water may have an influence onmoisture migration within the cheese during cooling.Temperature Induced Changes in Cheese pH and Moisture During Cooling

Cheese pH. Slabs of cheese were placed in an apparatus designed to movemoisture upward from position 1 to position 7 within a slab of cheese.Position 7 was the cold end position of the cheese and as the cheese isgradually raised out of the warm (27° C.) water over a period of 36 h,moisture migrates against the force of gravity from the bottom to thetop of the cheese slab. The temperature gradient in the slab of cheesemay also produce a gradient of pH within the slab of cheese. There was atrend (P=0.065) for cheese pH (Table 5) to increase from the bottom tothe top of the slab of cheese but the total range of difference in pH(i.e., 4.88 to 5.03) was small. No direct influence of CO₂ on cheese pHwas detected (Table 5), but there was a position by CO₂ treatment(P<0.01) interaction (Table 5).

TABLE 5 ANOVA (degrees of freedom, means squares, probabilities (inparenthesis) and R²) for CO₂ treatment, week of cheese making, andposition within cheese slab on percent moisture and pH of cheese exposedto a temperature gradient during cooling. Factor df Moisture pH Variabletype TR¹ 1 328.94  0.0004 Category (p < 0.01) (0.749)  W² 2 23.30 0.0068Fixed (p < 0.01) (p < 0.01) P³ 1 98.36 0.0373 Continuous (p < 0.01)(0.065)  TR × W 2  7.00 0.0028 (p < 0.01) (p < 0.01) P × W 2  0.030.0002  (0.547) (0.495)  P × TR 1 51.12 0.0131 (p < 0.01) (p < 0.01) P ×TR × W 2  0.57 0.0027 (p < 0.01) (p < 0.01) Error (for P, 30  0.0450.0003 P × TR, P × W, TR × W, and P × TR × W) R²  0.998 0.900  ¹TR =Treatment. ²W = Week. ³P = Position.Generally, there was almost no change in pH with position in the slabfor the control and there was a slight trend for pH to increase from thebottom to the top of the slab for the cheese made from milk with CO₂.The area of the slab of cheese that was cooled the fastest (i.e.,position 7) had the highest pH and the area of the slab that was cooledthe slowest (i.e., position 1) had the lowest pH.

Cheese moisture. There was a large impact (Table 5) of CO₂ treatment(P<0.01) and a significant position by CO₂ treatment interaction onmoisture migration in reduced fat Cheddar cheese (FIG. 4). The range ofmoisture migration across the seven positions within the slab of cheesefor the control was about 7.47%, while the moisture migration in thecheese made from milk containing CO₂ was approximately 1.3%. Clearly,the use of CO₂ reduced (ca. 80% reduction) the mobility of water duringthe cooling of the cheese and prevented the majority of moisturemigration. This result is even more impressive when one considers thefact that the moisture content of the reduced fat Cheddar made from themilk with added CO₂ contained 5.76% more moisture (Table 1) and had asignificantly higher moisture to protein ratio than the control (Table1). The reduction in the temperature induced moisture migration isconsistent with the large reduction in expressible serum in the cheesethat was produced by the addition of CO₂ to the milk prior to cheesemaking. Thus, it appears that addition of a sufficient quantity of CO₂to milk to reduce the pH to about 6.0 prior to cheese making, wouldsignificantly reduce moisture migration and the associated qualitydefects in 290 kg blocks of Cheddar cheese.

In some instances, the large increase in moisture due to CO₂ addition tomilk may not be desirable from a product characteristic point of view.During this study, no attempt was made to modify the cheese makingprocedure to produce cheese with lower moisture content from CO₂containing milk, but traditional modifications of the cheese makingprocedure should be able to bring the moisture content of cheese madefrom milk containing CO₂ closer to that of cheese made from milk withoutCO₂. However, if it was possible to carry a higher moisture level in thecheese and achieve acceptable cheese quality, then this would providesome yield benefit. This may be more feasible for higher moistureCheddar varieties that are not aged for long periods of time.

Thus, addition of hydrocolloids and denatured whey proteins to milkprior to cheese making increased cheese moisture content but did notreduce temperature induced moisture migration in Cheddar cheese duringcooling. Addition of CO₂ to milk prior to cheese making made a major(ca. 80%) reduction in temperature induced moisture migration inreduced-fat Cheddar cheese during cooling. The use of CO₂ also caused anincrease (ca. 5%) in cheese moisture in reduced-fat Cheddar cheese.Despite the higher moisture content, the temperature induced movement ofmoisture was still dramatically smaller for the reduced-fat Cheddarcheese made with CO₂ added to the milk. The CO₂ produced a reduction inmilk pH to about 5.9 to 6.0 and caused a movement of calcium out ofcasein micelles prior to rennet addition. The use of CO₂ in the milk didnot interfere with normal acid production by the starter culture and thefinal cheese pH was not influence by the addition of CO₂.

EXAMPLE 2 CO₂ Cheese Making Procedures Materials and Methods

Experimental Design and Statistical Analysis

Two 18 kg blocks of full-fat Cheddar cheese were. One block was madefrom milk with added CO₂ and another control block was made from milkwithout added CO₂. Cheese was made on three different days (using adifferent batch of milk each day) over a one-week period. A one-wayANOVA was used to determine if there was an impact of CO₂ on thecomposition and yield of the cheese. The least significant difference(P≦0.05) test was used to compare treatment means if the F-test for thestatistical model was significant (P≦0.05). The PROC GLM procedure ofSAS was used for all data analysis (SAS version 8.02, 1999-2001, SASInstitute Inc., Cary, N.C.)

Milk Processing and Cheese Manufacture

Milk processing and cheese manufacture were completed on the same day.Each day, raw whole bovine milk was received from the Cornell Universitydairy farm. The raw whole milk was pasteurized with a plate heatexchanger at 72° C. and a holding time of 15 sec. The pasteurized milkwas then cooled to 4° C. utilizing the regeneration and cooling sectionsof the system. Carbonated milk was collected after about 250 kg ofpasteurized milk was collected for the control treatment. A stainlesssteel sparger (7 μm) was inserted in-line after the cooling section ofthe pasteurization system. An additional 8 m of 2.54-cm diameterstainless steel pipe, with a sanitary conical-seat flow controllingvalve at the end, were added after the point of CO₂ injection to allow15 s of holding time for CO₂ incorporation into the cold pasteurizedmilk. A pressure gauge was located in-line at the point of injection andanother just before the flow control valve. Carbonation conditions (CO₂flow at 1.13 m³/h with 172 kPa back pressure at the flow control valve)were equilibrated with water (16.6 L/min) before milk was startedthrough the pasteurization system. Milk was carbonated to approximately1650 ppm to achieve a CO₂ level of about 1600 ppm in the cheese vat,which produced a milk pH of 5.9 at 31° C. The pH of 5.9 is between thetwo pH levels reported by Metzger et al. (2001) where increased solublenitrogen levels were observed.

All weights for cheese making including milk, whey, salt whey, cheese,and samples were weighed to the nearest gram (Model PE24, MettlerInstrument Co., Highstone, N.J.). After pasteurization, approximately240 kg of milk was weighed and placed into the control vat. The secondvat was filled with about 240 kg of pasteurized and carbonated milk.Cheese making for these two vats was conducted simultaneously. The milkin each vat was heated to 31° C., with agitation (Model 4MX; KuselEquipment Co., Watertown, Wis.). The milk was ripened for 45 min at 31°C. after the starter culture (911 DVS pellets, Chr. Hansen Inc.,Milwaukee, Wis.) was added at a concentration of 0.27 g/kg of milk. Whenripening was complete, annatto color (AFC WOS 550, Rhodia Inc., MadisonWis.) was added (0.0033 mL/kg of milk) to each vat. The ripened milk,31° C., was coagulated with double strength chymosin (0.1 mL/kg of milk;Chymax Extra, Chr. Hansen Inc., Milwaukee, Wis.). The chymosin wasdiluted, in 200 mL of water processed by reverse osmosis, immediatelybefore addition to the milk. After 30 min, the coagulum was cut (1.2 cmwire knives) and the curds and whey were not stirred for 5 min. After 5min, the curds and whey were stirred gently without added heat for 10min. The temperature was increased from 31 to 33° C. over 15 min andthen from 33 to 38° C. over an additional 15 min. The curds and wheywere continuously stirred and a temperature of 38° C. maintained untilthe target whey draining pH of 6.35 was attained. When the whey wasdrained, the curds were piled and allowed to knit together for 15 min.The large slab of curd was cut into two smaller slabs then turned. Thetwo curd slabs were stacked after 15 min. Curd slabs were maintained at38° C., piled two high, and turned over every 15 min throughout theCheddaring process. Curd slabs were milled when the curd pH reached5.30. Salt was added at 2.7% of the curd weight. The salt was dividedequally into three portions. The milled curds were dusted with a smallamount of the first portion of salt, then stirred for 2 min and allowedto sit for 10 min. The remainder of the first portion of salt was thenadded, the curds stirred, and then the curds were allowed to sit for 10min. The curds were salted with the two other portions of salt in 10 minintervals. The salted, milled curds were placed in a 18 kg capacitystainless steel Wilson hoop and pressed in an A-frame press (Model AFVS,Kusel Equipment Co., Watertown, Wis.) for 30 min at 70 kPa. Pressing wascontinued overnight, about 17 h, at 420 kPa. The cheese blocks werevacuum packaged and placed in a 4° C. cooler for 24 h before beingplaced in a cooler set at 6° C. for aging.

Sampling and Sample Preparation

Sampling. Raw whole milk at 4° C. was mixed and sampled immediatelybefore pasteurization. Pasteurized control and CO₂ treated milks werecollected after heating in the cheese vat to 31° C. prior to starteraddition. The whey collected from the start of curd draining to the endof draining was placed in a separate vat for each treatment and sampledfor CO₂ analysis. Additional whey collected throughout Cheddaring wasadded to the vats containing the whey. When all the whey from each vatwas collected, the whey was heated to 38° C. and mixed to assure uniformcomposition before a sample was taken for compositional analysis andused in mass-balance calculations. Salt whey was collected and weighedseparately after milling at the vat and mixed with the press whey, whichwas weighed. Press whey was collected during pressing by placing thehooped curds in large 8-mil plastic bags (model number S-5851, Uline,Waukegan, Ill.). Hot water was run on the outside of the bags to liquefyfat that may have solidified on the inside surface of the bag duringpressing and all of the whey was removed from the bags. A 1-cm thick by28 cm by 19 cm cross sectional slice from the center of the rectangular18-kg block of cheese was removed immediately after the block wasremoved from the press. This slice of cheese was used for compositionalanalysis and was vacuum-packaged and cooled to 4 C prior to analysis.

Sample preparation. Liquid samples were placed in 59-mL snap lid vialsand either analyzed fresh or stored frozen at −40° C. Frozen liquidsamples were thawed in a microwave oven in a manner that kept the sampletemperature below 10 C. Cheese slices were cut into 2-cm pieces thenground (Model 31BL92, Waring, New Hartford, Conn.) in 2 to 3 mm piecesand packed into 59-mL snap lid vials (Capital Vial, Inc., Fultonville,N.Y.) with no head space and either analyzed fresh or held frozen at−40° C. before analysis. Frozen cheese samples were thawed overnight at4° C. prior to analysis.

Standard Plate, Coliform, and Somatic Cell Counts

Standard plate and total coliform counts of pasteurized whole milks weredetermined by standard methods (Marshall, 1992; 6.2 and 7.8). SomaticCell Counts (SCC) of raw whole milk (AOAC 2000; 17.13.01, 978.26) weredetermined using a fluorimetric method (Milk-Scan Combi 4000, IntegratedMilk Testing; A/S N. Foss Electric Hillerød, Denmark) by a New YorkState licensed commercial laboratory (Dairy One, Ithaca, N.Y.).

Chemical Analyses

Milk, whey, and salt whey composition. Fat, total salt (TS), totalnitrogen (TN), nonprotein nitrogen (NPN), noncasein nitrogen (NCN)content of the milk, whey, and salt whey were determined using etherextraction (AOAC, 2000; 33.2.26, 989.05, forced air oven drying (AOAC,2000; 33.2.44, 990.20), Kjeldahl (AOAC, 2000; 33.2.11, 991.20), Kjeldahl(AOAC, 2000; 33.2.12, 991.21), Kjeldahl (AOAC, 2000; 33.2.64, 998.05),respectively. Crude protein (CP) was calculated by multiplying totalnitrogen by 6.38. The calcium content was determined using atomicabsorption (Metzger et al., 2000). CO₂ content of the milk and whey wasdetermined (Ma et al., 2001) using a CO₂ analyzer (MOCON Pac Check 650,MOCON, Minneapolis, Minn.). The Volhard method (Marshall, 1992; 15.5.B)was used to determine the salt content in the salt whey, using a 0.5-gtest portion. Milk, whey and salt whey compositions were determined intriplicate with the exception of calcium and CO₂, which were determinedin duplicate.

Cheese composition and pH. Fat content was determined using the Babcockmethod (Marshall (1992) at 15.8.A). Cheese moisture was determinedgravimetrically by drying 2 g of cheese in a forced-air oven at 10° C.for 24 h (AOAC, 2000; 33.2.44, 990.20) using 2 g of cheese. Salt contentwas determined using the Volhard method (Marshall, 1992; 15.5.B). TheKjeldahl method employing 1 g of cheese, was used to determine totalnitrogen (Lynch et al., 2002). The cheese calcium content was determinedby atomic absorption (Metzger et al., 2000).

Cheese pH was measured using a Xerolyt combination electrode (ModelHA405; Mettler Toledo, Columbus, Ohio) and an Accumet pH meter (Model AR25, Fisher Scientific, Pittsburgh, Pa.) after tempering to 23° C. Allanalyses were carried out in duplicate except total nitrogen, moisture,and fat which were performed in quadruplicate.

Component Recoveries

Fat, crude protein, calcium, total milk solids, and added saltrecoveries were determined by multiplying the weights (determined to thenearest g) of milk, whey, salt whey, and cheese by the compositionsdetermined by chemical analysis then dividing by the total weight ofeither fat, crude protein, calcium, total milk solids, or added salt andmultiplying by 100. Total milk solids recovery calculations did notinclude salt in the salt whey or in the cheese. If the mean actual totalunadjusted recoveries between treatments for the component were notsignificantly different (P>0.05), then the recoveries were adjusted bydividing the actual recoveries by the mean total recovery for each dayof cheese making and multiplying by 100.

Yield and Yield Efficiency

Actual cheese yields were calculated by ((cheese weight+curd sampleweight)/(milk weight−milk sample weight)×100. Moisture and salt adjustedyield was calculated accordingly (actual yield×(100−(cheese moisturecontent+cheese salt content)))/(100−(37+1.5)). The moisture and saltadjustment allows for comparison between treatments. Cheese yieldefficiency was calculated by dividing the adjusted yield by thetheoretical yield and multiplying by 100. Both Van Slyke and Barbanotheoretical cheese yield formulas (Neocleous et. al., 2002) were usedfor a cheese yield efficiency calculation.

The Van Slyke cheese yield formula for Cheddar cheese was calculatedaccording to the following formula: yield=(((0.93×percent fat inmilk)+(percent casein in the milk−0.1))×1.09)/(1−(target cheesemoisture/100)). The Barbano formula for Cheddar cheese differs from theVan Slyke in that the nonfat solids of the whey were used to determinethe nonfat whey solids retained in the water phase of the cheese(Barbano, 1996). The Barbano formula is useful when manufacturing apreacidified cheese because it can compensate for the loss of calciuminto the whey. Theoretical Cheddar cheese yield using the Barbanoformula was calculated using the following formula:yield=(A+B+C)/(1−((target cheese moisture+target cheese salt)/100))where A=(0.93×percent fat in milk), B=(percent casein inmilk−0.1)×(calcium phosphate retention factor), C=((((A+B)/(1−(actualcheese moisture percent/100)))−(A+B))×(percent nonfat wheysolids/100))×(solute exclusion factor). The calcium phosphate retentionfactors used in this study for the control and CO₂ treatments were 1.092and 1.082, respectively. The same calcium phosphate retention factor forthe control theoretical yield calculation was used by Neocleous et al.(2002). The lower calcium phosphate retention factor used for CO₂treatment theoretical yield calculation was obtained by plotting calciumretention factor data (acetic acid treatments) of Metzger et al. (2000)and using the second order polynomial equation (solute exclusionfactor=−0.0333(x²)+0.4333(x)−0.316) to compute a calcium retentionfactor to use for the milks (mean milk pH of 5.93) with added CO₂, wherex=milk pH. The solute exclusion factor of 0.6941 used by Neocleous etal. (2002) was also used in this study for both the control and addedCO₂ theoretical yield formulas.

Results

Milk Composition and Quality

Milk composition data are presented in Table 6. The mean casein as apercentage of true protein (TP) was about 83%, which in turn gave riseto a mean casein to fat ratio of 0.66. The pH of the milk at 31° C.before CO₂ addition (Table 6) was normal for good quality milk. Thestandard plate counts of the control and preacidified milks of 440 and1150 cfu/mL were low and not significantly different. The coliform countwas <10 cfu/mL for both treatments. The mean SCC of the milks used eachday for cheese making are shown in Table 6.

TABLE 6 Pasteurized control milk composition and pH for each day ofcheese making. Day of cheese making Component 1 2 3 Fat, % 3.44 3.423.35 Crude Protein, % 2.90 2.92 2.93 Non-Casein Protein¹, % 0.66 0.670.66 Nonprotein Nitrogen², % 0.20 0.18 0.19 TS, % 11.88 11.75 11.74Casein/Total Protein³, % 82.96 82.26 82.99 Casein/fat, % 0.65 0.66 0.68Calcium, % 0.105 0.106 0.106 pH⁴ 6.63 6.68 6.64 SCC, per mL 268,000260,000 226,000 ¹NCN = noncasein nitrogen × 6.38. ²NPN = NPN × 6.38.³CN/TP = (((CP − NCN)/((TN − NPN) × 6.38)) × 100). ⁴Measured at 31° C.

The CO₂ levels of the control and CO₂ treated milks were significantlydifferent after CO₂ addition (Table 7). The target milk pH of about 5.9was achieved (Table 8) by the addition of about 1600 ppm of CO₂ (Table7).

TABLE 7 Mean (n = 3) carbon dioxide content (ppm) of the control and CO₂treated milk and whey. Control CO₂ treated ppm LSD SEM Milk 88^(b)1615^(a) 77 19.7 Whey at draining 85^(b) 1000^(a) 115 29.4 ^(a,b)Meanswithin a row that do not share a common superscript differ (P ≦ 0.05).LSD = least significant difference; SEM = standard error mean.

TABLE 8 Mean (n = 3) milk, whey, and curd pH during cheese making.Control CO₂ LSD SEM Milk 6.65^(a) 5.93^(b) 0.054 0.014 Coagulantaddition¹ 6.57^(a) 5.93^(b) 0.052 0.013 Drain² 6.35^(a) 5.96^(b) 0.0740.019 Mill³ 5.30 5.30 NS 0.000 ^(a,b)Means within a row that do notshare a common superscript differ (P ≦ 0.05). ¹Ripened (45 min) milksample; ²Whey sample; ³Curd sample; LSD = least significant difference;SEM = standard error mean.Effect of CO₂ on Cheese Making

The mean (n=3) temporal patterns of pH for the control and CO₂ treatmentduring cheese making are shown in FIG. 4. The milk pH before starterculture addition, at coagulant addition, and of the whey at draining washigher (P≦0.05) for the control than the CO₂ treatment (Table 8). Theslope of the control pH curve from 0 to 120 min (FIG. 4) was negativewhereas the CO₂ treatment pH was constant over the same period. Thedownward slope of the control pH curve was expected because the starterculture was producing lactic acid during the 45 min of ripening.Although the starter culture growing in the milk of the CO₂ treatmentwas producing lactic acid, the pH of the whey did not change much fromthe initial pH of the milk until after about 130 min into cheese makingbecause the milk was also losing CO₂. The time from coagulant additionto whey draining (Table 9) was shorter for the CO₂ treatment (P<0.05).

Moreover, if CO₂ produced the milk pH decrease usually caused by lacticacid during ripening, then the lactic acid content of the sweet wheycollected at draining would be reduced. The CO₂ remaining in the wheycould be removed with a vacuum chamber. This might improve the qualityof whey products in certain applications. The separate impacts of lacticacid and CO₂ on the pH observed for the CO₂ treatment (FIG. 4) cannot bedetermined from the data collected.

The total make time was shorter with the milk preacidified with CO₂(Table 9) mostly due to the decrease in cooking time before wheydraining. The target whey draining pH of 6.35 at the end of cooking wassurpassed by the CO₂ treatment when the curd reached the final cookingtemperature (38° C.), while the control required an additional 10 min ofstir-out time at 38 C before the target pH was achieved (Table 9). Thecurds from the CO₂ treatment and control were both milled at pH 5.3.There was no significant difference in time from drain to mill betweenthe control and CO₂ treatment (Table 9). A shorter (P<0.05) total maketime of the CO₂ treatment was also reported by St-Gelais et al. (1997)using CO₂ to preacidify milk to pH 6.56 prior to cheese making, however,they reported a similar time from cut to whey draining and a 30 minshorter Cheddaring time.

TABLE 9 Mean (n = 3) Cheddar cheese make times for control and CO₂treated cheeses. Control CO₂ minutes LSD SEM Coagulant addition to drain88^(a) 78^(b) 2.6 0.67 Drain to mill 88  83  NS 1.72 Total 176^(a) 161^(b)  8.6 2.20 Stir out 10^(a)  0^(b) 0.0 0.00 ^(a,b)Means within arow that do not share a common superscript differ (P ≦ 0.05).

The effect of CO₂ was visually apparent during cheese making.Preacidification with CO₂ produced a firmer coagulum that could bedetected by touch and by the resistance of the coagulum when cut withwire knives. Other investigators have observed more rapid coagulation(Montilla et al., 1995; St-Gelais et al., 1997) and Van Slyke et al.(1903) observed that the rennetability of milk that had been heattreated above 85° C. was restored with the addition of CO₂ to milk priorto cheese making. After cutting, the curds of the CO₂ treated materialfloated to the surface with increasing temperature during cookingwhereas the curds of the control settled to the bottom if stirring wasnot constant. Floating curds may require a change in procedure if aportion of the whey is normally drained from the cheese vat through anoutlet located about half-way between the surface of the whey and thebottom of the vat (i.e., predraining). In the case of floating curds,predraining could be accomplished by draining a portion of the wheythrough the outlet located at the bottom of the cheese vat. Ifhorizontally stirred cheese vats were used to manufacture cheese frommilk containing CO₂, the thickness of the floating curd mass may be anissue that needs to be investigated with regard to curd integrity andfines. The fact that curds can be made to float in this process providesan opportunity to think about a different design of cheese vat and curdhandling system that could reduce curd shattering.

Whey, Salt Whey, and Cheese Composition

Whey and salt whey composition. A major portion of the CO₂ added to themilk was removed with the whey at draining (Table 7). Means andcomposition differences of whey and salt whey due to CO₂ treatment arereported in Table 10. CO₂ treatment resulted in a higher (P<0.05) fatcontent in the whey and salt whey. The calcium content was higher(P<0.05) in the whey from the CO₂ treatment and calcium content in thesalt whey was lower (P<0.05) than the control. There was no significantdifference in whey CP content but there was a slight increase in the CPcontent of the salt whey. There was substantially less salt in the saltwhey of the CO₂ treatment than the control (Table 10). St-Gelais et al.(1997) reported higher (P<0.05) fat content in the control whey versusthe CO₂ treatment (0.54 vs. 0.35%, respectively). The level of fat inwhey reported by St-Gelais et al. (1997) would normally be associatedwith much lower fat recovery in cheese (Barbano and Sherbon, 1984) anddoes not seem consistent with the high fat recovery in the cheese (91.93and 98.54%, respectively) reported in the same paper. The same authorsdid not detect a significant difference in the ash content of the whey,and salt whey composition was not reported.

TABLE 10 Mean (n = 3) whey, salt whey, and cheese composition. ControlCO₂ treated Component percent LSD SEM Whey Fat 0.27^(b) 0.37^(a) 0.0580.015 CP¹ 0.85 0.86 NS 0.020 NPN² 0.25 0.25 NS 0.005 TP³ 0.60 0.60 NS0.019 TS 6.71^(b) 6.87^(a) 0.098 0.025 Calcium 0.05^(b) 0.07^(a) 0.0020.0004 Salt whey Fat 1.28^(b) 4.07^(a) 1.174 0.299 CP 1.08^(b) 1.10^(a)0.023 0.006 NPN 0.40 0.40 NS 0.008 TP 0.68^(b) 0.71^(a) 0.024 0.006 TS16.15 15.73 NS 0.210 Salt 8.73^(a) 6.73^(b) 0.856 0.218 Calcium 0.21^(a)0.16^(b) 0.023 0.006 Cheese Fat 34.19^(a) 32.83^(b) 1.024 0.261 FDB⁴54.12^(a) 52.67^(b) 0.859 0.219 CP 23.72 24.02 NS 0.213 PDB⁵ 37.49 38.09NS 0.423 Moisture 36.84 37.67 NS 0.285 MNFS⁶ 55.97 56.08 NS 0.246 Salt1.44^(b) 2.24^(a) 0.119 0.030 Salt-in-the- 3.92^(b) 5.96^(a) 0.385 0.098moisture Calcium 0.69^(a) 0.52^(b) 0.035 0.009 Calcium/CP (×100)2.91^(a) 2.15^(b) 0.185 0.047 ^(a,b)Means within a row that do not sharea common superscript differ (P ≦ 0.05). ¹CP = crude protein (CP = totalnitrogen × 6.38). ²NPN = nonprotein nitrogen × 6.38. ³TP = true protein(TP = CP − NPN). ⁴FDB = fat on a dry basis. ⁵PDB = protein on a drybasis. ⁶MNFS = moisture in the nonfat substance.

Ultrafiltration is often used to fractionate whey before spray drying.Whey proteins, α-lactalbumin in particular, have been implicated inmembrane fouling (Tong et al., 1989). In addition to protein, solublecalcium has been found to reduce flux (Ramachandra Rao et al., 1994).With respect to fouling due to protein, there was no significantdifference in crude protein content of the whey (Table 10) between thecontrol and the CO₂ treatment. The calcium content of whey from the CO₂treatment (Table 10) was higher (P<0.05). Experiments can determine ifthe whey from CO₂ preacidification differs in ultrafiltration flux fromthat of typical Cheddar cheese whey. From the current literature it wasunclear whether the higher calcium content of the whey from the CO₂treatment would impact the process of dehydration during spray drying,the rehydration of whey powder, or functionality of the whey proteinconcentrate compared to the control.

Cheese composition and pH. No difference (P<0.05) in cheese crudeprotein, protein-on-a-dry basis, moisture, or moisture-in-the-nonfatsubstance was detected between the control and CO₂ treatment. The fatcontent of the control cheese was higher (P<0.05) than the CO₂ treatedcheese (Table 10). The CO₂ treatment cheese contained less calcium(Table 10) due to the reduced milk pH prior to rennet addition (Table8). The calcium content of the control was similar to the value of0.721%, standard error was 10.770, listed in the UDSA National NutrientDatabase (USDA, 2003), but the CO₂ treatment calcium content was lower.Additional experiments can determine if the lower calcium content of theCO₂ treatment cheese could reduce calcium lactate crystal formationduring aging. The control cheese pH, 5.00, was lower (P≦0.05) than theCO₂ treatment cheese pH, 5.09. The largest difference (P≦0.05) betweenthe control and treatment cheeses was salt content. The control cheesehad a salt content of 1.44% compared to 2.24% for the CO₂ treatment.Thus, the salt-in-the-moisture content for the CO₂ treatment (5.96%) washigher than the typical value (about 4.6%) for aged Cheddar. This couldimpact enzymatic changes during aging.

Component Recoveries

The actual total recoveries (i.e., accountability) for all componentswere not influenced by the CO₂ treatment. Actual total calcium, crudeprotein, fat, milk solids, and added salt recoveries for cheeses madefrom milk without and with added CO₂ were 101.91 and 102.13%, 101.89 and101.39%, 100.60 and 99.11%, 99.94 and 99.24%, 96.54 and 99.81%,respectively. Therefore, the actual recoveries were adjusted asdescribed in the materials and methods section of this paper (see Table11).

TABLE 11 Adjusted mean (n = 3) calcium, fat, CP, milk solids, and addedsalt recovery in whey, salt whey, and cheese. Control CO₂ percent LSDSEM Calcium recovery Whey 37.27^(b) 54.28^(a) 1.502 0.382 Salt whey3.55^(a) 2.44^(b) 0.362 0.092 Cheese 59.07^(a) 43.38^(b) 1.669 0.425 Fatrecovery Whey 6.98^(b) 9.79^(a) 1.708 0.435 Salt whey 0.68^(b) 1.89^(a)0.391 0.100 Cheese 93.08^(a) 87.57^(b) 1.921 0.489 CP recovery Whey25.48 25.65 NS 0.448 Salt whey 0.66 0.60 NS 0.030 Cheese 74.11 73.51 NS0.462 Milk solids recovery Whey 50.55^(b) 52.00^(a) 1.132 0.288 Saltwhey 1.15 1.24 NS 0.069 Cheese 48.65^(a) 46.41^(b) 1.281 0.326 Saltrecovery Salt whey 55.28^(a) 37.62^(b) 4.981 1.269 Cheese 43.04^(b)64.05^(a) 5.748 1.464 ^(a,b)Means within a row that do not share acommon superscript differ (P ≦ 0.05).

Calcium recovery. More (P<0.05) calcium was recovered in the whey of themilk treated with CO₂ (Table 11) than in the control (54.28 vs. 37.27%,respectively). Mean calcium recoveries were higher (P<0.05) in thecontrol salt whey and cheese compared to the CO₂ treatment (Table 11).Adding CO₂ to milk lowers the pH (Table 8) and causes an increase incalcium and phosphate concentrations in the serum phase of milk (Law andLeaver, 1998). The higher milk serum calcium content at coagulantaddition was the likely cause for the firmer coagulum of the CO₂treatment and the higher calcium content of the CO₂ treatment whey. Thelower calcium recovery in the cheese reduced cheese calcium content from0.69 to 0.52% (Table 10) was also due to the increased soluble calciumat coagulant addition.

Fat recovery. CO₂ treatment cheeses had a lower (P<0.05) fat recovery inthe cheese than the control cheeses (Table 11). Almost 10% of the totalmilk fat was recovered in the whey from the CO₂ treated milk compared toabout 7% in the control. The fat recovery for the control was consistentwith the assumption of 93% fat recovery in the Cheddar cheesetheoretical yield formulae described in the material and methodssection. The mean fat recovery in the salt whey of the CO₂ treatment wasmore than twice that of the control (Table 11). Although the differencein fat content between the control and the CO₂ treatment was greater forthe salt whey than the whey (Table 10), more fat was lost in the wheythan in the salt whey, as shown in Table 11.

The lower pH of the milk with CO₂ added prior to rennet addition changesthe rate and firmness of milk coagulation. The coagulation for the milkwith added CO₂ was faster and firmer in this study. Johnson et al.(2001) varied coagulation firmness in a controlled study of compositionand yield of 50% reduced fat Cheddar cheese and found an increase in fatloss with increased coagulation time and firmness at cut. Johnson et al.(2001) indicated that a coagulation that is cut soft will lose less fatand serum than a coagulation that is cut firm, if sufficient time isallowed for formation of the skin on the surface of curd particles aftercutting and before stirring. In the present study, the curd for the CO₂treatment was firmer than the control, but also was much lower in boundcalcium content.

Why was less fat recovered in the CO₂ treatment cheese than in thecontrol cheese? The milk pH of the CO₂ treatment (5.9) was closer to theacid pH optimum for chymosin. Higher enzyme activity may lead toexcessive casein hydrolysis at coagulation rather than specific actionon K-casein. Nonspecific proteolysis of casein by chymosin would reducethe casein structure's ability to hold fat and higher fat losses wouldbe observed. However, no significant difference in whey crude proteincontent or crude protein recovery in the cheese was detected between thecontrol and CO₂ treatment.

It is more likely that the lower calcium recovery, not caseinhydrolysis, in the cheese played a role in the lower fat recovery in thecheese of the CO₂ treatment. Increasing the milk serum calcium level byCO₂ treatment is similar to adding CaCl₂, in that both produce a morefirm milk coagulum. The end result of the two methods may be similar,but their impact on cheese composition differs. Adding CaCl₂ to milk(0.01 to 0.02% w/w) would not be expected to decrease the bound calcium.On the other hand, acidifying milk (i.e., adding CO₂ to a milk pH of5.9) would decrease bound calcium and colloidal phosphorus (Law andLeaver, 1998) and increase soluble calcium. Thus, the bound calcium andprobably the colloidal phosphorus content of the curd in the presentstudy were lower than if the coagulum would have been formed with addedcalcium. Also, the bound calcium and phosphorus were probably lower inthe CO₂ treatment than the control cheese indicated by the highercalcium content in the whey of the CO₂ treatment. The lower calciumcontent may have altered the ability of the curd to retain fat duringcooking, Cheddaring, salting, and pressing. Further work can identifythe exact point in time and the cause for the higher fat loss in thewhey when CO₂ is used to decrease the milk pH to 5.9 for the manufactureof full-fat Cheddar cheese and this will aid in development ofstrategies to reduce fat loss during manufacture of full-fat Cheddarwhen CO₂ is used in cheese making.

Crude Protein and milk solids recovery. No differences (P>0.05) weredetected in mean crude protein recoveries between the treatments for thewhey, salt whey, or cheese (Table 11). Total milk solids recovery didnot include added salt only milk solids. Total milk solids recovery ofthe CO₂ treatment was higher in the whey and lower in the cheese. Thedifferences in milk solids recovery were generally consistent with thedifferences in fat and calcium recovery due to CO₂ treatment.

Salt recovery. An unexpected result of the current study was thedifference (P≦0.05) in salt recovery in the cheese between the controland CO₂ treatment (Table 11). About 64% of the added salt was recoveredin the cheese of the CO₂ treatment compared to only 43% in the controlcheese. The large difference (Table 10) in salt content between controland CO₂ cheeses occurred even though the curd-salting rate was the samefor both treatments. No difference (P>0.05) was detected in cheesemoisture (Table 10), but the CO₂ treatment caused thesalt-in-the-moisture to be one and a half times that of the control.St-Gelais et al. (1997) did not detect a difference in cheese saltcontent between the control and CO₂ treatment. The CO₂ treatment ofSt-Gelais et al. (1997) not only had a higher milk pH at coagulantaddition (6.47), but a higher curd pH at salting (5.46). In our study,the coagulant and salt were added at lower pH values for the CO₂treatment than in the work of St-Gelais et al. (1997). It is unclearwhether the lower calcium content of the curd at salting was responsiblefor the high salt uptake of CO₂ treatment cheese. A marked improvementof added salt retention in Cheddar cheese, like the results shown inTable 11, would reduce salt wastes from a cheese manufacturing facility.

Cheese Yield and Yield Efficiency

Actual and adjusted cheese yields were significantly lower for the CO₂treatment (Table 12). The Van Slyke theoretical yield formula predictedthe same yield for both the control and the CO₂ treatment because themilk compositions were the same (Table 12).

TABLE 12 Mean (n = 3) actual, moisture and salt adjusted, Van Slyke andBarbano theoretical cheese yields and cheese yield efficiencies. ControlCO₂ LSD SEM Yield kg/100 kg Actual 9.26 9.07 NS 0.052 Adjusted¹ 9.29^(a)8.86^(b) 0.302 0.077 Van Slyke 9.21 9.21 NA² NA Barbano 9.21 9.18 NA NAYield efficiency percent Van Slyke 100.9^(a) 96.2^(b) 2.599 0.662Barbano 100.9^(a) 96.5^(b) 2.760 0.703 ^(a,b)Means within a row that donot share a common superscript differ (P ≦ 0.05). ¹Moisture adjusted to37% and salt to 1.5%. ²NA = not applicable.Cheese yields predicted by the Barbano theoretical yield formula for theCO₂ treatment cheeses were lower because of the different calciumphosphate retention factor used for the CO₂ treatment that allowed forthe expected lower retention of calcium phosphate in the cheese. Thecheese yield efficiency of the control was 100.9%. The mean fat recoveryattained with the control cheese (Table 11) was consistent with thetheoretical fat recovery (93%) of the theoretical yield formulae, whichindicates that the cheese making methods alone did not create the fatloss observed in the CO₂ treatment cheese. Preacidification with CO₂resulted in a 4.7% lower Van Slyke yield efficiency and a 4.4% lowerBarbano yield efficiency than the control. The difference in yieldefficiency within the CO₂ treatment (0.3%) represents the reduction inyield due to mineral loss. The fat loss in the whey caused a greaterdifference in yield efficiency between the control and treatment thancalcium loss.

These results indicate that cheese manufactured from milk acidified to apH of 5.9 using approximately 1600 ppm of CO₂ retained less calcium andfat than the control cheese. The higher loss of fat was primarily in thewhey at draining. Preacidification with CO₂ did not alter the crudeprotein recovery in the cheese. The CO₂ treatment resulted in a higheradded salt recovery in the cheese and produced a cheese that containedmore salt than needed. Considering the higher added salt retention ofthe CO₂ treatment, the salt application rate can be lowered to achieve atypical cheese salt content. Cheese yield efficiency of the CO₂ treatedmilk was 4.4% lower than the control due to fat loss. The use of CO₂ ledto several beneficial effects including better milk coagulation, reducedrennet use, less need for salt addition and potentially reduced problemswith calcium crystal formation. Moreover, the CO₂ procedure led toproduction of a reduced fat cheese. While such a reduction in fat isdesirable in many instances, fat loss can be eliminated if desired bychanges in the manufacturing procedure.

EXAMPLE 3 Less Water Migration and More Uniform Moisture Content isObserved in Cheese Made from Milk Preacidified with CO₂

Experimental Design

One 18-kg block of milled-curd Cheddar cheese (35×29×19 cm) wasmanufactured per treatment (from milk with added CO₂ and without addedCO₂) on three different days.

Pasteurized whole milk was carbonated to approximately 1600 ppm CO₂,which resulted in a milk pH at the vat of 5.93 compared to 6.65, at 31°C., for the control. Cheese manufacturing conditions were kept constantfor the two treatments with the exception the whey from the milk withadded CO₂ was drained at pH 5.96 compared to 6.35 for the control. Theaddition of CO₂ decreased the total manufacturing time because of theshorter stir-out time. The usage rates of chymosin and salt were thesame for both treatments. Cheeses were pressed overnight (17 h). Whenthe cheeses were removed from the press the temperature in the center ofthe blocks was about 29° C. A more detailed description of cheese makingconditions is described in Example 1.

The CO₂ content, titratable acidity (TA), pH, soluble nitrogen andcasein degradation of the cheeses were monitored over 6 mo of aging at6° C. Changes in the water phase (monitored by analysis of expressibleserum (ES)) were determined.

Sampling and Sample Preparation

Unsalted milled curd (USMC) and cheese sampling. Unsalted milled curdsamples were taken after milling at pH 5.3, placed in plastic bags, andimmediately prepared for removal of expressible serum (ES). Cheeses weresampled by removing three cross sections of cheese, with approximatedimensions of 1 cm by 28 cm by 19 cm, from the center of the blockimmediately after the block was removed from the press. The first crosssection was vacuum packaged for compositional analysis. The second crosssection was used for the expressible serum procedure. The sides of thethird section were trimmed to leave a center piece of about 9 cm by 15cm, which was vacuum-packaged and used for CO₂ analysis. After the threeslices were removed from the center of the block, the two remainingpieces of the block were placed into a plastic bag and vacuum packagedfor further aging. Sampling was done again at approximately 30, 90, and180 d.

Sample preparation of USMC and cheese. Cheeses and unsalted milled curdwere cut into 2-cm pieces, ground (Model 31 BL92, Waring, New Hartford,Conn.) into 2 to 3 mm, and packed into 59-mL snap lid vials leaving nohead space and either analyzed fresh or they were frozen at −80° C. andheld until the time of analysis. Cheese slices for CO₂ analysis were notground, but were cut into approximately 3-mm pieces immediately beforeanalysis.

Expressible Serum preparation. Expressible serum from unsalted milledcurd and cheese immediately after pressing was collected at 25° C. asdescribed in Guo and Kindstedt (1995), except that the samples werecentrifuged at 23,500×g. Expressible serum from several centrifugebottles for each cheese treatment was combined to obtain a enough samplefor chemical analyses. Expressible serum was placed in 59-mL snap-topvials and frozen at −80° C.

Chemical Analyses

Expressible Serum composition. Total nitrogen (TN) content of theexpressible serum was determined in duplicate using the Kjeldahl method(AOAC, 2000; 33.2.11, 991.20). Crude protein was calculated bymultiplying the total nitrogen by 6.38. Calcium content was determinedin duplicate by atomic absorption (Metzger et al., 2000).

USMC and cheese composition and pH. The fat content was determined byBabcock method (Marshall, 1992; 15.8.A). Moisture was determinedgravimetrically by drying in a forced-air oven at 100° C. for 24 h(AOAC, 2000; 33.2.44, 990.20) using a 2-g cheese test portion. Saltcontent was determined using the Volhard method (Marshall, 1992;15.5.B). The Kjeldahl method (1-g test portion) was used to determinetotal nitrogen (Lynch et al., 2002) and crude protein was calculated(TN×6.38). Fat and salt content were not determined for unsalted milledcurd. Cheese pH was determined using a Xerolyt combination electrode(Model HA405; Mettler Toledo, Columbus, Ohio) with an Accumet pH meter(model AR 25, Fisher Scientific, Pittsburgh, Pa.) after tempering to 23°C. Titratable acidity (TA) (AOAC, 2000; 33.7.14, 920.124) of the cheesewas determined as described by Lau et al. (1991). All analyses werecarried out in duplicate except total nitrogen and fat, which wereperformed in quadruplicate.

CO₂ content of Milk and Cheese. A method of standard additions (MOSA)was used to determine the CO₂ content of cheese by a modification of themethod described by Ma et al. (2001) for determining the CO₂ content ofmilk. The MOSA was selected because the control cheeses contained asmall background level of CO₂ (thus, no blank matrix was available) andbecause the technique is especially useful when an analyte (e.g., CO₂)is present in low concentrations near the limit of quantification, whichwas the case for the control cheeses. In the MOSA, the sample is testedinitially and with increasing added amounts of the analyte, essentiallycreating a calibration curve using the sample itself (Miller, 1991;González et al., 1999)

For the initial CO₂ determination, cheese was cut into approximate 3-mmcubes and 20±0.1 g was weighed into a small, stainless steel blenderassembly (catalog number 14-15-18B, mini-sample container, 37-110 mLcapacity, Fisher Scientific, Pittsburgh, Pa.). Then, 20 mL of degassedreverse osmosis purified water and 10 mL of 1 N sulfuric acid wereadded. The blender was immediately covered with Parafilm M (PechineyPlastic Packaging, Chicago, Ill.) and tightly secured with a rubberband. The contents of the jar were blended at low speed for 30 s thenfor 15 s at 1-min intervals for a total of 5 blends over a 5 min period.At 15 s after the last blend, a sticky nickel (catalog number 380-035,MOCON, Minneapolis, Minn.) was placed on the Parafilm M cover. The CO₂content in the headspace was determined by sampling with a gas-samplingneedle inserted through the sticky nickel, taking care to keep theneedle out of cheese slurry. The sampling needle was connected to aninfrared CO₂ analyzer (Pac Check 650, MOCON, Minneapolis, Minn.)previously calibrated with room air (“0” CO₂) and 99.8% CO₂ (catalognumber 23402, manufactured for Supelco, Bellefonte, Pa., by ScottSpecialty Gases). A reading of the CO₂ content (percent CO₂) of theheadspace was then taken. After the initial reading was obtained, thesame procedure was repeated 5 times using a new 20 g portion of the samecheese sample each time. CO₂ levels were increased in 30 to 50%increments over the previous reading. CO₂ was added to the sample bydecreasing the amount of degassed water initially added and substitutinga corresponding volume of sodium bicarbonate standard solution (0.5g/100 g, equivalent to ca 2.6 mg CO₂/g or 2600 ppm CO₂) so that thefinal amount of added standard solution and degassed water totaled 20mL.

A MOSA linear regression equation (y=mx+b) was constructed from theinitial and 5 determinations with added sodium bicarbonate standardsolution, where y=instrument reading (% CO₂), m=slope, x=sodiumbicarbonate added (expressed as ppm CO₂ in cheese) and b=intercept. Theconcentration of CO₂ in an individual cheese was calculated byextrapolation of the regression equation to y=0 and using the absolutevalue of x at y=0. Visual inspection of the experimental data and theresulting coefficients of determination (R²≧0.99) indicated theresulting regression equations were linear.

Proteolysis

Cheese pH 4.6 and 12% TCA soluble nitrogen as a percentage of totalnitrogen (SNPTN) were determined in duplicate as described by Bynum andBarbano (1985). SDS-PAGE was performed as described by Neocleous et al.(2002) except a 7 μL sample (1 g of cheese per 10 mL of sample buffer)was loaded per lane for all cheese samples and a constant 15%concentration acrylamide gel was used. Results of the SDS-PAGE analysiswere reported as the ratio of α-casein and β-casein to para-κ-casein.This was done to normalize the data for small variations in sampleloading that can result from sample preparation since para-K-casein isnot hydrolyzed during aging (Nath and Ledford, 1973). The ratios ofα_(s)-casein and β-casein to para-κ-casein were used by Lau et al.(1991) and Neocleous et al. (2002) in the casein degradationcalculations that were reported by those investigators. However, Lau etal. (1991) and Neocleous et al. (2002) used those ratios to calculatethe percentage of casein degraded in the cheeses by using the first dayof analysis as 0% of casein degraded. In those studies there was nodifference between treatments at time zero. As a result, the data fromthe first day of analysis was not reported. In the experiments reportedherein, the first day of analysis was very important because of thedifferences between treatments immediately after the cheeses wereremoved from the press. During aging α_(s)-casein and β-casein werehydrolyzed and their bands on the SDS-PAGE gel became less intense,while para-κ-casein remained constant. A decreasing ratio indicatesproteolysis of either α_(s)-casein or β-casein.

Expressible serum from cheese and unsalted milled curds were preparedusing 0.9 mL of the sample buffer containing dithiothreitol as describedby Verdi et al. (1987) and 0.1 mL of expressible serum. SDS waspurchased from Sigma-Aldrich Chemical (L-4390; St. Louis, Mo.). A 10 to20% SDS-PAGE gradient gel (Verdi et al., 1987) was used for expressibleserum electrophoresis. Unsalted milled curd expressible serum gels wereloaded with 16 μL of sample plus buffer per lane for both the controland CO₂ treatment. Cheese expressible serum loadings of sample plusbuffer for the control and CO₂ treatment were 8 μL and 4 μL,respectively because the expressible serum from the CO₂ treatmentcontained more protein than the control expressible.

The presence of α_(s1)-I-casein in the expressible serum of the CO₂treatment cheeses was determined by an additional experiment where anα_(s)-casein solution and a milk were separately incubated withchyrnosin. Samples were then analyzed with our SDS-PAGE procedure. After1 hour of incubation a large protein band was present below theα_(s1)-casein in the β_(s)-casein solution treated with chymosin. Aftertwo hours that band was more pronounced. A band in the same location waspresent in the milk sample after incubation with chymosin. The bandappeared below the β-casein. Since α_(s1)-casein was found by Creamerand Richardson (1974) to be the primary proteolytic product of chymosinaction on α_(s1)-casein, this unknown band in the expressible serum ofthe CO₂ treatment was likely α_(s1)-I-casein. The presence of theα_(s1)-I-casein band after the β-casein band in the gels run asdescribed herein differed from those in report of Malin et al. (1995).Protein migration patterns can be different due to different sources ofSDS (Swaney et al., 1974). Although Malin et al. (1995) did not reporttheir source of SDS, different sources of SDS were the likely cause ofthe α_(s1)-I-casein migration differences.

Statistical Analysis

The PROC GLM procedure of SAS was used for all data analysis (SASversion 8.02, 1999-2001, SAS Institute Inc., Cary, N.C.). The leastsignificant difference test (P<0.05) was used to compare treatment meansof the compositional data if the F-test for the statistical model wassignificant (P<0.05). One-way ANOVA was used to analyze cheese and USMCcomposition data. For comparison of the control and CO₂ treatment at anyone sampling period (i.e. 0, 30, 90, and 180 d) a t-test was performed.ANOVA was used to analyze data over the aging period and least-squaremeans are reported in the text for CO₂ content, titratable acidity, pH,SNPTN, α_(s)-casein:para-κ-casein, and β-casein:para-κ-casein of thecontrol and CO₂ treatment cheeses over the 6 mo of aging. Age wasanalyzed as a continuous variable. A mathematical transformation of theage variable was necessary to minimize multicollinearity of the linearand quadratic forms of the age variable (Glantz and Slinker, 2001). Thetransformation of age, age=day of storage at 6° C.−((last testingday−first testing day)/2), made the data set orthogonal with respect toage. The quadratic term for age and the interaction of age by treatmentwere included in the statistical model if significant, or if notsignificant to show that the curvature was not detected, in the case ofCO₂ content during aging (FIG. 5).

Results

USMC and Cheese Composition

No difference (P>0.05) was detected between the control and CO₂treatment for unsalted milled curd moisture and crude protein (Table13). As expected, the calcium content of the USMC was lower for the CO₂treatment (Table 13) because of the lower pH at draining (5.96 vs.6.35). No difference in CP, PDB, moisture, and MNFS was detected betweenthe control and CO₂ treatment cheeses (Table 13). The fat content andfat on a dry basis were higher (P≦0.05) for the control cheese. Thelower (P≦0.05) calcium content in the CO₂ treatment cheese was expected,but the higher (P≦0.05) salt content (Table 13) of the CO₂ treatmentcheese was not expected.

TABLE 13 Mean (n = 3) unsalted milled curd (USMC) and Cheddar cheesecomposition. Control CO₂ Component percent LSD SEM USMC Moisture 46.5846.35 NS 0.834 Crude Protein 20.29 20.92 NS 0.273 Calcium 0.63^(a)0.46^(b) 0.063 0.016 Calcium/Crude Protein (×100) 3.09^(a) 2.20^(b)0.344 0.088 Cheddar cheese Fat 34.19^(a) 32.83^(b) 1.024 0.261 FDB¹54.12^(a) 52.67^(b) 0.859 0.219 Crude Protein 23.72 24.02 NS 0.213 PDB²37.49 38.09 NS 0.423 Moisture 36.84 37.67 NS 0.285 MNFS³ 55.97 56.08 NS0.246 Salt 1.44^(b) 2.24^(a) 0.119 0.030 Salt-in-moisture 3.92^(b)5.96^(a) 0.385 0.098 Calcium 0.69^(a) 0.52^(b) 0.035 0.009 Calcium/CrudeProtein (×100) 2.91^(a) 2.15^(b) 0.185 0.047 ^(a,b)Means within a rowwithout a common superscript differ (P ≦ 0.05). ¹FDB = fat on a drybasis. ²PDB = protein on a dry basis. ³MNFS = moisture in the nonfatsubstance.

The least squares mean CO₂ content of the treatment cheese (337 ppm) washigher (P≦0.01) than the control (124 ppm) and did not change duringaging (Table 14, FIG. 5). The least squares mean pH of the control,4.98, was lower (P≦0.01) than the treatment, 5.14. A linear age bytreatment interaction was detected as well as a quadratic function ofage (P≦0.01, Table 9, FIG. 6).

TABLE 14 Type III SS for cheese CO₂, pH, titratable acidity (TA),soluble nitrogen as a percentage of total nitrogen (SNPTN), and ratiosof α_(s)-casein and β-casein to para-κ-casein at 0, 30, 90, 180 d ofaging. 12% α_(s)- β- pH 4.6 TCA CN:para- CN:para- Factor df CO₂ pH TASNPTN SNPTN κ-CN κ-CN Treatment¹ (T) 1 273494** 0.15** 0.14** 30**  2**2** 0.1 Age (A) 1 3668 <0.01 0.61** 914**  236**  16**  3.3** A × A 13686 0.02** — 59** 10** 5** — A × T 1 — 0.01** 0.03* — — 1*  — Error 19— 0.02 — — — 3  — Error 20 27160  — 0.13 8  3  — — Error 21 — — — — — —1.9 R²     0.91 0.89 0.85   0.99   0.99  0.88 0.64 *P ≦ 0.05. **P ≦0.01. ¹Treatments are cheeses manufactured from milk with and withoutadded CO₂.The least squares mean titratable acidity of the control cheese, 1.01%,was higher than (P≦0.01) than the CO₂ treatment cheese, 0.87%, and wasconsistent with the difference in pH (FIGS. 5 and 6). The titratableacidity, increased as a linear function of cheese age (Table 9, FIG. 4)and there was an age by treatment interaction with the titratableacidity of the control cheese increasing faster with age than the CO₂treatment cheese (FIG. 7).Proteolysis

The CO₂ treatment had higher (Table 14, P≦0.05) mean levels of pH 4.6and 12% TCA soluble nitrogen as a percentage of total nitrogen (SNPTN)than the control immediately after pressing, 6.44% versus 4.79% and2.71% versus 2.03%, respectively (FIG. 8). During aging, the CO₂treatment had a higher (P≦0.01) least squares mean content of pH 4.6SNPTN, 15.31%, than the control, 13.08%. The CO₂ treatment alsocontained more (P≦0.01) 12% TCA SNPTN, 6.85%, than the control, 6.28%,during aging. The pH 4.6 and 12% TCA SNPTN increased in both the controland CO₂ treatment over the 6 mo aging period (FIG. 7) both as a linearand a quadratic function of age (Table 14, P≦0.01). The level of pH 4.6and TCA SNPTN were similar to levels reported previously for Cheddarcheese (Lau et al., 1991; Neocleous et al., 2002.)

No difference (P>0.05) in α_(s)-casein:para-κ-casein andβ-casein:para-κ-casein ratios were detected between the unsalted milledcurds of the control and CO₂ treatment, data not shown. The differencein the α_(s)-casein:para-κ-casein ratio between control and CO₂treatment cheeses was more pronounced at 0 d when the cheeses wereremoved from the press (FIG. 9) than at any other time. When the cheeseswere removed from the press the CO₂ treatment had a lower (P≦0.05)α-casein:para-κ-casein ratio than the control 2.48 and 3.87,respectively. The least squares mean α_(s)-casein:para-κ-casein ratio ofthe CO₂ treatment cheese, 1.26, was lower (P≦0.01) than the controlcheese, 1.88, during 6 mo of aging (FIG. 9). Theα_(s)-casein:para-κ-casein ratio changed both as a linear and quadraticfunction of age and there was a linear age by treatment interaction(Table 14). No significant difference (P>0.05) in β-casein:para-κ-caseinratio was detected between the control and CO₂ treatment immediately outof the press or during aging (FIG. 9, Table 14). The linear function ofage was significant (Table 14), because the β-casein:para-κ-casein ratiodecreased in both the control and CO₂ treatment cheeses during the agingperiod.

USMC and Cheese Expressible Serum

No difference (P>0.05) in the amount of unsalted milled curd expressibleserum was detected between the control and CO₂ treatment (Table 8).There was a large decrease in the amount of expressible serum for boththe control and CO₂ treatment due to salting and pressing. After saltingand pressing, almost twice the amount of expressible serum could beremoved from the control cheese compared to the CO₂ treatment (Table 8).Expressible serum from the CO₂ treated unsalted milled curds had aslightly higher (P≦0.05) CP content than the control. After salting andpressing the crude protein content of the expressible serum from the CO₂treatment cheese was much higher than the control (Table 15). Thecalcium content of the unsalted milled curd and cheese expressible serumfrom the CO₂ treatment was lower than the control. Because the crudeprotein was higher and calcium was lower in the expressible serum of theCO₂ treatment, the calcium expressed as a percentage of crude proteinwas much lower (P<0.05) than the control for both the unsalted milledcurd and cheese. Neither the CO₂ treatment nor the control unsaltedmilled curd expressible serum contained a detectable amount of casein onan SDS-PAGE gel (data not shown). Casein was found in the CO₂ treatmentcheese expressible serum but not in the control cheese expressible serum(FIG. 9).

As shown in this Example, using milk preacidified with CO₂ led todecreased water migration and a more uniform cheese moisture content.Moreover, less rennet and salt is needed when milk is preacidifiedbefore cheese making. The CO₂ content of Cheddar cheese manufacturedusing milk preacidified with CO₂ was consistently higher during agingthan the control cheese. However, such increased CO₂ content was notdetrimental and actually provided certain benefits. Increased CO₂content can inhibit microbial growth, thereby increasing cheese shelflife. Hence, using preacidified milk for cheese making provides severalbenefits.

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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “an antibody” includes a plurality (forexample, a solution of antibodies or a series of antibody preparations)of such antibodies, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed:
 1. A method for making a uniformly moist cheese thatcomprises: acidifying pasteurized milk to a pH of about 5.6 to about 6.2at a temperature of about 88° F. to about 105° F. with carbon dioxide toproduce an acidified milk culture, and making a uniformly moist cheesetherefrom; wherein the cheese has about 4% to about 20% less fat than acheese made without carbon dioxide acidification.
 2. The method of claim1, wherein the milk is acidified to a pH of about 5.9 to about 6.0 at atemperature of about 90° F. to about 100° F.
 3. The method of claim 1 ,wherein the pasteurized milk is acidified with about 1000 ppm to about3500 ppm carbon dioxide.
 4. The method of claim 1 , wherein thepasteurized milk is acidified with about 1400 ppm to about 1800 ppmcarbon dioxide.
 5. The method of claim 1, wherein the method furthercomprises adding a starter culture of lactic acid producing bacteriaafter acidifying the pasteurized milk.
 6. The method of claim 1, whereinthe method further comprises coagulating the pasteurized milk with acoagulating agent.
 7. The method of claim 6, wherein the coagulatingagent is rennet.
 8. The method of claim 7, wherein less rennet is usedthan when the same amount of milk is not acidified.
 9. The method ofclaim 6, wherein the rennet employed in the method is about half therennet used when the same amount of milk is not acidified.
 10. Themethod of claim 6, which further comprises cutting coagulate formed bycoagulating the pasteurized milk with the coagulating agent to therebyform a whey-curd suspension.
 11. The method of claim 10, which furthercomprises removing some whey from the whey-curd suspension after heatingthe whey-curd suspension to about 99-103° F, cooling the removed wheyand adding the cooled whey to the heated whey-curd suspension when thewhey-curd suspension reaches a pH of about 5.4 to about 5.8.
 12. Themethod of claim 1, wherein the method further comprises adding salt tocurds formed while making cheese.
 13. The method of claim 12, whereinless salt is added than would have been added to curds made from milkthat has not been acidified.
 14. The method of claim 1, wherein thecheese formed by the method is pressed into blocks.
 15. The method ofclaim 14, wherein the cheese is cut into particles or shredded.
 16. Themethod of claim 1, wherein the cheese formed by the method is not aged.17. The method of claim 1, wherein the cheese formed by the method isfrozen without aging.
 18. The method of claim 1, wherein the methodfurther comprises aging the cheese.
 19. The method of claim 18, whereinflavor in the cheese develops faster during aging than in cheese madefrom milk that is not acidified.
 20. The method of claim 1, wherein thecheese produced is American, Cheddar, Monterey Jack, mozzarella,Muenster, or Swiss.
 21. The method of claim 1, wherein the cheeseproduced is a low fat or reduced fat cheese.
 22. A method of makingcheese comprising carbonating pasteurized whole milk to about 1500 ppmCO₂ to about 1800 ppm CO₂ and initiating a cheese making procedure;wherein the cheese has about 4% to about 20% less fat than a cheese madewithout carbon dioxide acidification.